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Stereoselective assay of tocainide enantiomers and study of their selective disposition in man Pillai, Gopalakrishna 1984

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STEREOSELECTIVE ASSAY OF TOCAINIDE ENANTIOMERS AND STUDY OF THEIR SELECTIVE DISPOSITION IN MAN by GOPALAKRISHNA PILLAI Pharra., B i r l a In s t i tu te of Technology & Science, 1967 M . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Facul ty of Pharmaceutical Sciences) D i v i s i o n of Pharmaceutical Chemistry We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ju ly 1984 © Gopalakrishna P i l l a i , 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requ i rements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree tha t , the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copy ing of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s unders tood t h a t copy ing or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l lowed w i t h o u t my w r i t t e n p e r m i s s i o n . F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s Department o f 22 The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3 August 17, 1984 - i i -ABSTRACT Toca in ide , a s t r u c t u r a l analog of l i d o c a i n e , i s an o r a l l y e f f e c t i v e antiarrhythmic agent. The chemical s t ructure of toca in ide includes an asymmetric center and the drug i s used c l i n i c a l l y i n the racemic form. Although the antiarrhythmic propert ies of the toca inide enantiomers have not been studied i n man, the R(-) enantiomer i s three times more potent than the S(+) isomer as an antiarrhythmic agent i n a mouse model. Pharmacokinetic studies of enantiomers can reveal whether the s t e r e o s e l e c t i v i t y l i e s i n the process of absorpt ion , d i s t r i b u t i o n , metabolism or a combination of these processes. In order to study t h e i r d i s p o s i t i o n a very s e n s i t i v e and s e l e c t i v e a n a l y t i c a l technique i s required that i s capable of r e so lv ing the two isomers when present together i n b i o l o g i c a l f l u i d s . The main ob jec t ive of t h i s study was to develop such a method and to apply i t for the simultaneous measurement of toca in ide enantiomers i n human plasma, ur ine and s a l i v a and to examine t h e i r s t e reose lec t ive d i s p o s i t i o n . A gas chromatographic method using a f u s e d - s i l i c a c a p i l l a r y column coated with a c h i r a l s ta t ionary phase ( C h i r a s i l - V a l ® ) was developed for the d i r e c t r e s o l u t i o n of toca in ide enantiomers. Base- l ine r e s o l u t i o n of t h e i r hepta f luorobutyryl der iva t ive s was achieved and no i n t e r f e r i n g peaks were observed i n plasma, ur ine or s a l i v a ex t rac t s . The i d e n t i t y of the resolved peaks was es tabl i shed by comparison of the r e t en t ion time, o p t i c a l r o t a t i o n and mass spectra of toca in ide enantiomers, which were obtained by a s t e reospec i f i c - i i i -synthes i s . In order to study s t e reose lec t ive d i s p o s i t i o n , 200 mg of ( ± ) toca inide hydrochlor ide ( t ab le t s ) were given o r a l l y to seven healthy male subjects and by intravenous in fu s ion to f i v e of them. Blood (8 mL) was withdrawn at predetermined time in te rva l s up to 72 hours and urine was c o l l e c t e d up to 96 hours fo l lowing drug admin i s t ra t ion . S a l i v a (2 mL) samples were a lso c o l l e c t e d at the time of blood c o l l e c t i o n . C a l i b r a t i o n curve data and p r e c i s i o n of assay of plasma, ur ine and s a l i v a were determined by t r i p l i c a t e analyses of s ix concentrat ions ranging from 50 ng to 3000 ng of ( ± ) toca inide hydrochlor ide along with 1000 ng of 1-aminoacetoxylidide (W-49167) as the i n t e r n a l standard. The r e l a t i v e standard devia t ions were i n the range of 4 to 9.9% for plasma, 2.6 to 7.2% for urine and 2.1 to 5.9% for s a l i v a . The lowest concentrat ion that could be determined by the s p l i t mode of i n j e c t i o n was 25 ng of each enantiomer per mL of plasma. The plasma concentrat ion-t ime data were analysed by a computer program (AUTOAN and NONLIN) and were found to f i t a two compartment model. The h a l f - l i v e s ca l cu la ted from the plasma data , fo l lowing an o r a l dose, were 16.3 ± 4 hours for the dextro-isomer and 11.9 ± 2.7 hours for the levo-isomer. The corresponding h a l f - l i v e s fo l lowing an intravenous dose were 17.0 ± 2.5 hours and 11.7 ± 2.4 hours r e s p e c t i v e l y . The plasma clearances were 144 ± 28 mL/min and 222 ± 54 mL/min for the dextro- and levo-isomers , r e s p e c t i v e l y . The volumes of d i s t r i b u t i o n , (V<j)3, were 2.47 ± 0.43 L/Kg and 2.52 ± 0.49 L/Kg for the dextro- and levo-isomers r e s p e c t i v e l y . Fol lowing o r a l admin i s t r a t ion , both isomers were absorbed r a p i d l y at the same rate and peak l e v e l s were reached at the same time. The area under - i v -the plasma concentrat ion-t ime curve fo l lowing o r a l dose was higher than that fo l lowing intravenous adminis t ra t ion and therefore the b i o a v a i l a b i l i t y was 151 ± 43% for the dextro- isomer and 167 ± 67% for the levo-isomer. B i o a v a i l a b i l i t y ca lcu la ted from urine data was 109 ± 18% for the dextro-isomer and 112 ± 29% for the levo- i somer . The mean enantiomer r a t i o , (+) t oca in ide / ( - ) t oca in ide , i n the plasma was 1.52 at 24 hours and 1.88 at 48 hours fo l lowing an o r a l dose of the racemate. S imi lar enantiomer r a t i o s were observed i n the ur ine but enantiomer composition i n the s a l i v a was d i f f e ren t i n that the levo-isomer l e v e l was higher than the dextro- isomer and that the l e v e l s of both the isomers were higher than the corresponding plasma concentrat ions . The sa l iva/plasma concentrat ion r a t i o was 2.06 ± 0.50 for the dextro-isomer \ and 3.68 ± 0.76 for the levo-isomer. The c o r r e l a t i o n between s a l i v a and plasma concentra t ion , ranged from, r = 0.910 to 0.987 for the dextro-isomer and r = 0.884 to 0.986 for the levo-isomer. Tocainide enantiomer d i s p o s i t i o n was also studied i n a pat ient with renal dysfunct ion (serum crea t in ine = 13.8 mg%). The h a l f - l i f e of the dextro-isomer was 45 hours and that of the levo-isomer was 28.7 hours , which corresponded to a 2.5 fo ld increase as compared to heal thy sub ject s . The plasma clearance was 86 mL/min and 146 mL/min for the dextro- and levo-isomers , r e s p e c t i v e l y . During hemodialys i s , the h a l f - l i f e was decreased to 6.5 hours and 5.4 hours for the dextro- and levo- i somers , r e s p e c t i v e l y . Thus i t can be concluded that the d i s p o s i t i o n of toca inide enantiomers i n the human i s s t e reose lec t ive and that the enantiomer r a t i o i n the plasma i s v a r i a b l e between subjects . - v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES x i i i LIST OF FIGURES x v i SYMBOLS AND ABBREVIATIONS xx ACKNOWLEDGEMENTS x x i i I . INTRODUCTION 1 A. Plasma Concentrat ion Monitoring of Antiarrhythmic Drugs 1 B. Pharmacokinetics of Tocainide 4 1. Absorption and Plasma Concentrations 7 2. Intravenous Admini s t ra t ion 7 3. D i s t r i b u t i o n 8 4 . Metabolism and E l i m i n a t i o n 9 5. E f f ec t s of Disease on Pharmacokinetics 11 6. Plasma Concentrations and C l i n i c a l e f fects 12 7. Side e f fec t s 13 8. Drug Interact ions 15 9 . Comparison with other Antiarrhythmic Drugs 16 10. Methods of Ana lys i s of Tocainide i n B i o l o g i c a l F lu id s 16 C. S tereose lec t ive Drug Analys i s 20 1. Resolut ion of Diastereomers 20 2. D i r e c t Resolut ion of Enantiomers 21 - v i -Page 3. Resolut ion of Enantioraers by HPLC 22 4. S tereospec i f i c Radioimmunoassays 23 5. Resolut ion of Tocainide Enantiomers 24 D. Renal F a i l u r e and Drug Accumulation 25 E . Glucuronidat ion i n Renal F a i l u r e 29 F . E n a n t i 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 31 1. Enant io se lec t ive D i s t r i b u t i o n 31 2. Enant io se lec t ive Metabolism 33 3. Inversion of Conf igurat ion as a Metabol ic Process 34 G. Spec i f i c Aims of the Project 35 I I . EXPERIMENTAL 39 A. Mater ia l s and Chemicals 39 1. Column Chromatography 39 2. Thin- layer Chromatography 39 3. High-performance L iqu id Chromatography 39 4. G a s - l i q u i d Chromatography 40 5. Animal Surgery 40 6. Miscel laneous 41 B. Preparat ion of Reagents and Stock Solut ions 41 1. Tocainide Hydrochloride So lut ion 41 2. W-49167 Hydrochloride So lut ion 42 3. a-Bromonaphthalene So lut ion 42 4. Monoethylg lyc inexyl id ide Hydrochloride S o l u t i o n . . . 42 5. Tocainide Base So lut ion 42 6. Naphthoresorcinol Reagent 43 - v i i -Page C. Pre l iminary Studies on C a p i l l a r y Column Gas Chromatography of Tocainide 43 1. Column S e l e c t i o n . . . . 43 1.1 Preparat ion of Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column 43 1.2 G a s - l i q u i d Chromatographic (GLC) Ana lys i s of Tocanide on Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column 44 1.3 GLC Analys i s of Tocainide Using C a p i l l a r y Columns Coated with S i l a r 10 C, OV-225 and SP-2330 45 2. Determination of Optimum Condit ions for D e r i v a t i z a t i o n 45 3. Se l ec t ion of Interna l Standard 46 4. Check for S p l i t t e r D i f f e r e n t i a t i o n Between Tocainide and Interna l Standard, Monoethylg lyc inexyl id ide (MEGX) 46 D. Pharmacokinetics of Tocainide i n the Rat 47 1. Plasma Leve l Study 47 1.1 Preparat ion of Jugular Vein Cannula 47 1.2 Surg i ca l Implantation of the Cannula 48 1.3 Drug Admini s t ra t ion and S e r i a l Blood C o l l e c t i o n 49 2. Urinary Excre t ion Studies 50 E . Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Rat Plasma and Urine Assay 50 F . Resolut ion of Tocainide Enantiomers on t C h i r a s i l - V a l ® Glass C a p i l l a r y Column 51 G. Synthesis of Tocainide Enantiomers 52 1. Synthesis of R( - ) toca in ide hydrochlor ide 52 2. Synthesis of S(+)tocainide hydrochlor ide 53 - v i i i -Page 3. Determination of O p t i c a l P u r i t y and Ident i ty of R(-) and S(+) Tocainide by Gas Chromatography/Mass Spectrometry (GCMS) 54 4. Measurement of O p t i c a l Rotat ion of Tocainide Enantiomers 55 H. 1. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Assay of Tocainide Enantiomers i n Human Plasma and Urine 55 2. Inter- and Intra-assay Var ia t ions 55 I . Pre l iminary Study of S t e r e o s e l e c t i v i t y of Tocainide D i s p o s i t i o n i n the Humans 56 J . 1. Comparison of A n a l y t i c a l Results Obtained for Racemic Tocainide Using Carbowax 20 M Fused-s i l i c a C a p i l l a r y Column with those of Tocainide Enantiomers Using C h i r a s i l - V a l ® Glass C a p i l l a r y Column 57 2. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Human Plasma Assay by Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column 57 K. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n 58 1. Human Plasma and Urine Assay Using C h i r a s i l - V a l ® F u s e d - s i l i c a C a p i l l a r y Column and W-49167 as Internal Standard 58 2. Confirmation of the Structure of Acyl Der iva t ive of 1-Aminoacetoxylidide (W-49167) by C a p i l l a r y GCMS 58 L . The Ef fect of Sodium Hydroxide Treatment on Urine Containing Tocainide Carbamoyl-0-3-D-Glucuronide . . . . . 58 M. Pharmacokinetics of Tocainide Enantiomers i n Healthy Human Subjects 60 1. Intravenous Adminis t ra t ion of Racemic Tocainide Hydrochloride 60 2. Oral Admini s t ra t ion of Racemic Tocainide Hydrochloride 60 - ix -Page N. 1. Assay of Tocainide Hydrochloride Tablets 61 2. Assay of Tocainide Hydrochloride I n j e c t i o n 61 3. Determination of Adsorption of Tocainide by the P l a s t i c Tubing of the Infusion Set 62 0. 1. Chromatographic Analys i s of Uremic Plasma Extract on a C h i r a s i l - V a l ® F u s e d - s i l i c a C a p i l l a r y Column 62 2. Chromatographic Analys i s of Uremic Plasma Ex t rac t on a Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column 62 3. Chromatographic Analys i s of Uremic Plasma Extracts on a Dual C a p i l l a r y Column (Carbowax 20 M and C h i r a s i l - V a l ® ) 63 4 . Pre l iminary Study of Tocainide Enantiomer D i s p o s i t i o n i n an Anephric Pa t ient 63 4.1 Intravenous Admini s t ra t ion of Racemic Tocainide Hydrochloride 63 4.2 Hemodialysis 64 P. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Assay of S a l i v a 64 Q. Ana lys i s of Tocainide Metabol i tes i n Human Urine 65 1. I s o l a t i o n of Glucuronides from Urine by Adsorption on XAD-2 Resin Column 65 2. Th in- l ayer Chromatographic Separation of the Glucuronides 66 3 . Preparat ive T h i n - l a y e r Chromatographic I s o l a t i o n of Tocainide Glucuronides 66 4 . Microbore LCMS of Tocainide Glucuronides 68 5. I d e n t i f i c a t i o n of Band I b as Tocainide Carbamoyl-0-3-D-glucuronide 68 5.1 L i q u i d Chromatographic Ana lys i s of the Hydantoin Derived from the G l u c u r o n i d e . . . . . . 68 - x -Page 5.2 GC and GCMS Analys i s of the Hydantoin Derived from the Glucuronides 69 5.3 Acid Hydro lys i s of the Glucuronide to Tocainide Enantioraers 70 5.4 Enzyme Hydro lys i s of the Glucuronide to Tocainide Enantiomers 70 6. Gas Chromatography/Mass Spectrometry (GCMS) of Tocainide Glucuronides 71 6.1 Permethylation of Glucuronides 71 6.1.1 Preparat ion of Dry DMSO 71 6.1.2 Preparat ion of Dimsylsodium Carbanion 71 6.1.3 Permethylat ion 72 I I I . RESULTS AND DISCUSSION 74 A. G a s - l i q u i d Chromatographic Analys i s of Tocainide Enantiomers Using F u s e d - s i l i c a C a p i l l a r y C o l u m n s . . . . . 74 1. Se lec t ion of Sui table Interna l Standard 81 2. Measurement of S p l i t t e r D i f f e r e n t i a t i o n Between Tocainide and Interna l Standard, MEGX 82 3. Minimum Detectable Quantity 83 4. A p p l i c a t i o n of C a p i l l a r y Gas Chromatography for the Assay of Rat Plasma and Urine 83 5. Pharmacokinetics of Intravenous Tocainide i n the Rat 86 B. Resolut ion of Tocainide Enantiomers 89 1. I d e n t i f i c a t i o n of Resolved Peaks 95 2. C a l i b r a t i o n Data and P r e c i s i o n of Assay of Tocainide Enantiomers i n Human Plasma and U r i n e . . 98 3. Pre l iminary Study of S t e r e o s e l e c t i v i t y i n Tocainide D i s p o s i t i o n i n Man 103 4 . E f f e c t of Sodium Hydroxide Treatment on Urine Containing Toca in ide-carbamoyl-O-^D-Glucuronide . I l l - x i -Page C. Pharmacokinetics of Tocainide Enantiomers i n Man 118 1. Pharmacokinetics of Ora l Tocainide Enant iomers . . . 118 2. Pharmacokinetics of Intravenous Tocainide Enantiomers 121 2.1 Plasma Clearance 132 2.2 Renal Clearance 133 2.3 Volume of D i s t r i b u t i o n 133 3. Tocainide Enantiomer Levels i n the Urine 134 4 . Chromatographic Analys i s of Uremic Plasma Ext rac t on C h i r a s i l - V a l ® F u s e d - s i l i c a C a p i l l a r y Column 146 5. Pharmacokinetics of Tocainide Enantiomers i n Renal Dysfunction and During Hemodialysis 149 5.1 Pharmacokinetics of Intravenous Tocainide Enantiomers 152 5.2 D i s p o s i t i o n of Drug Glucuronides i n Renal F a i l u r e 157 5.3 Hemodialysis 158 5.3.1 Clearance Ca lcu la t ions During Hemodialysis 160 6. S tereose lec t ive Sa l ivary Excre t ion of Tocainide Enantiomers i n Man 163 6.1 Tocainide Enantiomers i n the S a l i v a of a Pa t i ent with Renal Dysfunction 172 D. Ana lys i s of Tocainide Metabol i tes i n the Urine 175 1. Gas Chromatographic Analys i s of Glucuronides 177 2. L i q u i d Chromatographic Analys i s of G l u c u r o n i d e s . . . 178 3. I s o l a t i o n of Glucuronides from Urine 179 4 . Microbore LCMS of Tocainide Glucuronides 182 5. I d e n t i f i c a t i o n of Band 1^ as Tocainide Carbamoyl-O-3-D-Glucuronide 184 - x i i -Page 6. Gas Chromatographic/Mass Spectrometric (GCMS) Ana lys i s of Permethylated Glucuronides 189 6.1 CI and EI Mass Spectra of Permethylated Glucuronic Acid 189 6.2 CI and EI Mass Spectra of Permethylated p-Ni trophenol Glucuronide 193 6.3 GCMS of Permethylated Tocainide Glucuronides Using an SE-30 F u s e d - s i l i c a C a p i l l a r y Column 193 6.4 GCMS of Permethylated Tocainide Glucuronides Using a C h i r a s i l - V a l ® F u s e d - s i l i c a C a p i l l a r y Column 201 SUMMARY AND CONCLUSIONS 204 REFERENCES 206 - x i i i -LIST OF TABLES Table Page 1 Area r a t i o s at d i f f e rent s p l i t r a t i o s . 83 2 C a l i b r a t i o n curve data and p r e c i s i o n of rat plasma and urine assay 87 3 Pharmacokinetic parameters of intravenous toca in ide i n the rats 88 4 Percent drug excreted unchanged i n the 24 hour rat ur ine 89 5 C a l i b r a t i o n curve data and p r e c i s i o n of assay of toca in ide enantiomers i n human plasma 101 6 C a l i b r a t i o n data and p r e c i s i o n of assay of toca in ide enantiomers i n human ur ine 102 7 Inter- and intra-sample v a r i a t i o n s i n area r a t i o s 105 \ 8 Plasma concentrat ion-time data fo l lowing an o r a l J dose of 3 mg/kg toca inide to two healthy subjects 106 \ 9 C a l i b r a t i o n curve data and p r e c i s i o n of assay of human plasma using carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column 107 10 H a l f - l i v e s of toca inide enantiomers and the raceraate . N\$*f i n two healthy subjects .„108-~7 ± ^ 11 C a l i b r a t i o n curve data and p r e c i s i o n of assay of plasma using C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column and W-49167 as Interna l standard 113 12 C a l i b r a t i o n curve data and p r e c i s i o n of assay of ur ine using C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column and W-49167 as i n t e r n a l standard 114 13 Plasma concentrat ion-t ime data fo l lowing an o r a l dose of 200 mg of ( ± ) toca in ide hydrochlor ide i n seven healthy volunteers 119 14 Pharmacokinetic parameters of toca inide enantiomers fo l lowing an o r a l dose of 200 mg of ( ± ) toca inide hydrochlor ide 120 - x iv -Page 15 Plasma concentrat ion-t ime data fol lowing an intravenous in fus ion of 200 mg of ( ± ) toca inide hydrochlor ide to f ive healthy volunteers 123 16 Enantiomer r a t i o , (+)T0C/(-)T0C, i n plasma fo l lowing an intravenous in fu s ion of 200 mg of ( ± ) toca inide hydrochloride to f ive healthy volunteers 124 17 Pharmacokinetic parameters of toca inide enantiomers fo l lowing an intravenous in fus ion of 200 mg of ( ± ) toca inide hydrochlor ide to f ive healthy v o l u n t e e r s . . . . 127 18 Pharmacokinetic parameters of toca inide enantiomers ( ca lcu la ted from AUC) fo l lowing an intravenous in fus ion of 200 mg of ( ± ) toca inide hydrochlor ide to f ive healthy volunteers 128 19 B i o a v a i l a b i l i t y (%) of toca inide enantiomers from plasma data 129 20 Assay of standard toca inide and of samples prepared from tablets and i n j e c t i o n s 131 21 Assay re su l t s before and after the i n j e c t i o n of toca inide hydrochlor ide i s passed through the i n f u s i o n set 132 22 Cumulative excret ion of toca in ide enantiomers i n the ur ine (%) fo l lowing o r a l and intravenous dose of 200 mg ( ± ) toca inide hydrochlor ide 135 23 Ur inary excret ion data for the rate p l o t : dose-i . v . in fus ion of 200 mg ( ± ) toca inide h y d r o c h l o r i d e . . . 140 24 Urine data for amount remaining to be excreted (ARE) p lo t of S(+) t o c a i n i d e : dose i . v . in fus ion of 200 mg ( ± ) tocainide hydrochlor ide 141 25 H a l f - l i v e s of tocainide enantiomers ca lcu la ted from plasma and urine data 142 26 Enantiomer r a t i o , (+)T0C/(-)T0C, i n the plasma and urine fol lowing an o r a l dose of 200 mg ( ± ) toca in ide hydrochlor ide to seven healthy volunteers 143 27 C a l i b r a t i o n data and p r e c i s i o n of uremic plasma assay using dual c a p i l l a r y column (carbowax 20 M and C h i r a s i l - V a l ® ) 151 - XV -Page 28 Plasma concentrat ion-time data fo l lowing an intravenous in fu s ion of 200 mg of ( ± ) tocainide hydrochlor ide to a pat ient with renal dysfunct ion 153 29 Pharmacokinetic parameters of tocainide enantiomers fo l lowing an intravenous in fus ion of 200 mg of ( ± ) tocainide hydrochlor ide to a pat ient with rena l dysfunct ion and during hemodialysis 156 30 A r t e r i a l and venous concentrat ions of the isomers of tocainide and the A-V di f ference 162 31 C a l i b r a t i o n curve data and p r e c i s i o n of assay of s a l i v a 165 32 Tocainide enantiomers i n the s a l i v a of healthy subjects fo l lowing an intravenous in fus ion of 200 mg of ( ± ) tocainide hydrochlor ide 166 33 Sal iva/plasma concentrat ion r a t i o s of toca inide enantiomers i n healthy volunteers 167 34 Enantiomer r a t i o , (+)T0C/(-)T0C, i n plasma, urine and s a l i v a fo l lowing intravenous in fus ion of 200 mg of ( ± ) tocainide hydrochloride to healthy volunteers 171 35 Comparison of observed and predicted sa l iva/plasma r a t i o of S(+) tocainide i n one of the healthy volunteers 173 36 Tocainide enantiomers i n the s a l i v a of a pat ient with renal dysfunct ion and sa l iva/plasma r a t i o s 176 37 R e l a t i v e i n t e n s i t i e s of fragment ions of permethylated g lucuronic acid i n the CI and EI mass spectra 194 38 Re la t ive i n t e n s i t i e s of fragment ions of permethylated p-ni t rophenol glucuronide i n the CI and EI mass spectra 196 39 Re la t ive i n t e n s i t i e s of fragment ions of permethylated glucuronides of tocainide extracted from urine 199 - x v i -LIST OF FIGURES Figure Page 1 Structures of toca inide and l i d o c a i n e 5 2 Chromatogram of p o l a r i t y mixture on carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column 76 3 Chromatogram of hepta f luorobutyry l d e r i v a t i v e of toca inide on carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column 78 4 Chromatograms of hepta f luorobutyryl d e r i v a t i v e of toca inide on S i l a r 10 C, 0V-225 and SP-2330 glass c a p i l l a r y columns 79 5 Time-dependence of d e r i v a t i v e formation 80 6 Structure of monoethylg lyc inexyl id ide (MEGX) 81. 7 ECD response of 3 picograms of toca in ide on carbowax 20 M f u s e d - s i l i c a c a p i l l a r y c o l u m n . . . 84 8 A. Rat plasma p r o f i l e 3 hours a f ter intravenous dose of toca in ide hydrochlor ide 85 B. Rat ur ine conta ining 1.0 ug each of toca in ide and i n t e r n a l standard (MEGX) 85 9 Structures of toca inide enantiomers 90 10 S tereose lect ive events p r io r to b i o l o g i c a l r e s p o n s e . . . . 91 11 Structure of C h i r a s i l - V a l ® s ta t ionary phase 93 12 Rat urine p r o f i l e 24 hours a f ter an intravenous dose of ( ± ) toca in ide hydrochlor ide on C h i r a s i l - V a l ® glass c a p i l l a r y column 94 13 Chromatogram of ( ± ) tocainide heptaf luorobutyrate on C h i r a s i l - V a l ® glass c a p i l l a r y column 96 14 Synthesis of R(-) toca inide 97 15 T o t a l ion-current p r o f i l e of hepta f luorobutyryl d e r i v a t i v e of R(-) toca inide 99 16 EI mass spectra of hepta f luorobutyryl de r iva t ive s of toca in ide enantiomers 100 - x v i i -Page 17 Plasma, ur ine and s a l i v a p r o f i l e 24 hours a f ter an o r a l dose of ( ± ) toca inide hydrochlor ide 104 18 Tocainide enantiomers: plasma with 0.2 ug of each enantiomer and 1.0 ug of i n t e r n a l standard on C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column 109 19 Structures of 1-aminoacetoxylidide (W-49167) and toca in ide 110 20 CI mass spectra of hepta f luorobutyryl d e r i v a t i v e of i n t e r n a l standard, W-49167 112 21 Tocainide enantiomers: plasma p r o f i l e 24 hours a f ter an o r a l dose of the racemate 115 22 Tocainide enantiomers: ur ine p r o f i l e 92 hours a f ter an o r a l dose of the racemate 116 23 Tocainide enantiomers: plasma concentrat ion-t ime curves a f ter an o r a l dose of the racemate 122 24 Tocainide enantiomers: plasma concentrat ion-t ime curves a f ter an intravenous in fu s ion of ( ± ) t o c a i n i d e . . 126 25 Cumulative amount of S(+) toca inide excreted i n the ur ine 137 26 Semilogarithmic p lot of excret ion rate versus time a f ter an intravenous adminis t ra t ion of ( ± ) toca in ide hydrochlor ide 138 27 Semilogarithmic p lo t of the amount remaining to be excreted versus time (ARE plot ) a f ter an intravenous dose of ( ± ) toca inide hydrochlor ide 139 28 Urine p r o f i l e before (A) and af ter (B) hydro ly s i s with 3-glucuronidase 145 29 GC/ECD p r o f i l e of uremic plasma blank on C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column 147 30 Chromatogram of blank uremic plasma extract containing 16.8 mg % c rea t in ine on carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column 148 31 GC/ECD p r o f i l e of uremic plasma blank 2 hours a f ter intravenous in fu s ion of ( ± ) toca in ide to an anephric p a t i e n t . A . C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column B. Carbowax 20 M and C h i r a s i l - V a l ® 150 - x v i i i -Page 32 Log plasma concentration-time curve fo l lowing an intravenous i n f u s i o n of 200 mg of ( ± ) tocainide hydrochloride to a pat ient with rena l dysfunct ion 155 33 Log a r t e r i a l plasma concentration-time curve during 5 hour hemodialysis 161 34 Tocainide enantiomers: s a l i v a p r o f i l e one hour after intravenous in fus ion of the racemate 168 35 Log plasma and s a l i v a concentrations vs time for R(-) and S(+) tocainide enantiomers 170 36 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 tocainide enantiomers fo l lowing intravenous in fu s ion of the racemate. . 174 37 I s o l a t i o n of tocainide glucuronides from human u r i n e . . . . 181 38 Microbore LCMS of tocainide glucuronides 183 39 Microbore LCMS of p-ni trophenol glucuronide 185 40 HPLC of hydantoin (standard) (A) and hydantoin derived from a glucuronide of tocainide (B) 186 41 GC of hydantoin (standard) (A) and hydantoin derived from a glucuronide of tocainide (B) 187 42 CI mass spectra of a hydantoin d e r i v a t i v e obtained from a glucuronide of tocainide 188 43 Chromatogram of the hepta f luorobutyryl d e r i v a t i v e s of tocainide enantiomers obtained by ac id -hydro ly s i s of band l b 190 44 GC/MS/CI of permethylated g lucuronic a c i d . Column: SE-30 f u s e d - s i l i c a c a p i l l a r y 191 45 Fragmentation pattern of permethylated g lucuronic a c i d . 192 46 GC/MS/CI of permethylated p-ni trophenol g lucuronide . Column: SE-30 f u s e d - s i l i c a c a p i l l a r y 195 - x ix -Page 47 GCMS of permethylated glucuronides of toca in ide . Column: SE-30 f u s e d - s i l i c a c a p i l l a r y 198 48 GC/MS/EI of permethylated glucuronides of toca in ide . Column: C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y 202 - X X -SYMBOLS AND ABBREVIATIONS a d i s t r i b u t i o n rate constant 3 d i s p o s i t i o n rate constant AUC area under the plasma l eve l - t ime curve CI chemical i o n i s a t i o n EI e l e c t r o n impact C l ^ plasma clearance C1 D renal clearance ( V d ) „ volume of d i s t r i b u t i o n P (Vd)gg volume of d i s t r i b u t i o n at steady-state V c volume of cen t ra l compartment K e ^ e l imina t ion rate constant k excre t ion rate constant e k non-renal excret ion rate constant nr K Q t dose given by intravenous in fus ion AUMC area under the f i r s t moment of the plasma l e v e l time curve cumulative amount excreted in the urine amount removed by d i a l y s i s Clpp d i a l y s i s plasma clearance C^ , concentrat ion i n a r t e r i a l plasma C„ concentrat ion i n venous plasma - x x i -Qp plasma flow rate through d i a l y s e r blood flow rate through d i a l y s e r H hematocrit (+)TOC S(+) toca in ide (-)TOC R(-) toca inide HFBA hepta f luorobutyr ic anhydride HPLC high-performance l i q u i d chromatography GLC g a s - l i q u i d chromatography ECD e l e c t r o n capture detector i . v . intravenous MEGX monoethylg lyc inexyl id ide W-49167 1-aminoacetoxylidide TOCG toca in ide carbamoyl-0-$-D-glucuronide DMSO dimethylsul foxide K 1 2 transport rate constant from compartment 1 to compartment 2 K 2 i t ransport rate constant from compartment 2 to compartment 1 - x x i i -ACKNOWLEDGEMENT The author wishes to acknowledge Dr . K e i t h McErlane fo r h i s guidance and supervi s ion throughout the course of t h i s i n v e s t i g a t i o n . The author i s a l so g ra te fu l to Dr . Jim Axelson fo r h i s constant support and h e l p f u l d i scuss ions and to Drs . Frank Abbott , Jim Orr and John P r i c e for t h e i r help and guidance during the course of t h i s study. A word of thanks go to Mrs. Barbara McErlane for' her ass i s tance during the chemical study, to Rowland Burt for h i s help i n obta in ing mass spec t r a l data , to David Kwok for h i s ass i s tance i n metabol i te i s o l a t i o n and l i q u i d chroma-tography and to Wayne Riggs and Ram K a p i l for t h e i r help during animal surgery. The f i n a n c i a l support provided by U n i v e r s i t y of B r i t i s h Columbia, As t ra Pharmaceuticals and B . C . Health Care Research Foundation i s g r a t e f u l l y acknowledged. - 1 -INTRODUCTION A. Plasma Concentration Monitoring of Antiarrhythmic Drugs The sub-optimum use of p o t e n t i a l l y e f f ec t ive drugs i s a frequent cause of therapeutic f a i l u r e . Marked 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 of pharmacokinetics and pharmacodynamic responses i n pat ients with otherwise s i m i l a r c h a r a c t e r i s t i c s may lead to inadequate therapeutic management or adverse react ions when a standard dose i s administered. Determination of plasma or serum drug concentrat ion i s , therefore , i n c r e a s i n g l y recommended to guide and adjust drug treatment i n var ious c l i n i c a l cond i t ions . Therapeutic monitoring of phenytoin, theophyl l ine and aminoglycoside a n t i b i o t i c s i s now w e l l es tabl i shed and carr ied out r o u t i n e l y i n many h o s p i t a l s . S imi l a r monitoring of important ant iarrhythmic agents i s now fea s ib le with the recent i n t r o d u c t i o n of rap id and s p e c i f i c enzyme immunoassays, high-performance l i q u i d chromatographic and gas chromatographic methods. In combination with e l e c t r o p h y s i o l o g i c a l t e s t ing and continuous ECG recordings , plasma concentrat ion measurements would s u b s t a n t i a l l y contr ibute to greater e f f i c a c y and safety of ant iarrhythmic treatment. Monitor ing of ant iarrhythmic drugs i n pat ients could g rea t ly f a c i l i t a t e opt imisa t ion of therapy i f the fo l lowing bas ic requirements are f u l f i l l e d : (1) A d i rec t r e l a t i o n s h i p between plasma concentrat ion and ant iarrhythmic and/or tox ic e f fects of the drug can be demonstrated. (2) Upper and lower l i m i t s of the therapeut ic - 2 -concentra t ion range are wel l de f ined . (3) Plasma concentrat ion p r o f i l e s at the recommended dosage are h ighly v a r i a b l e . Numerous c l i n i c a l s tudies ind ica te that present ly ava i l ab le ant iarrhythmic drugs f u l f i l l these formal reasons for plasma concentrat ion monitor ing . A d i r e c t in f luence of drug l eve l s on antiarrhythmic e f f i cacy and t o x i c i t y of qu in id ine was f i r s t reported by Sokolow and Edgar (1950). Subsequently, the existance of r e l a t i v e l y narrow therapeutic ranges were also reported for l i d o c a i n e (Giannel ly et a l . , 1967), procainamide (Koch-Weser and K l e i n , 1971), mexi let ine (Campbell, et a l . , 1973, Talbot et a l . , 1973), disopyramide (Niarchose, 1976), tocainide (Winkle et a l . , 1976; Winkle et a l . , 1978; Woosley et a l . , 1977; Ryans and K e r l i n e r , 1979), f l e c a i n i d e (Somani, 1980; Anderson et a l . , 1981), l o r c a i n i d e (Meinertz et a l . , 1979), verapamil (Eischelbaum et a l . , 1980; Johnston et a l . , 1981) and aprindine (Zipes et a l . , 1977; Greene et a l . , 1978). With a few exceptions, the margin between therapeutic and toxic doses of the ant iarrhythmic drugs i s r e l a t i v e l y smal l . Dosage adaptation i s often d i f f i c u l t s ince age, c a rd i ac , hepat ic or renal funct ion may inf luence the absorption, d i s t r i b u t i o n and e l imina t ion of these agents. In a d d i t i o n , drug in terac t ions with enzyme-inducing agents, such as phenytoin, phenobarbital or r i f a m p i c i n may also occur. I t i s therefore d i f f i c u l t to pred ic t whether a given dose w i l l achieve the des ired plasma concentrat ion i n i n d i v i d u a l pa t i ent s . Despite obvious i n d i c a t i o n s there are no c o n t r o l l e d studies ava i l ab le to prove that ant iarrhythmic treatment guided by drug l e v e l monitoring i s - 3 -d i s t i n c t l y super ior to a c a r e f u l l y se lected drug regimen based on c l i n i c a l judgment alone. Concurrent plasma concentrat ion obtained at the time of e l e c t r o p h y s i o l o g i c a l tes t ing may al low the c l i n i c i a n to i d e n t i f y a therapeutic concentrat ion for an i n d i v i d u a l pa t i ent . However, i f no c l i n i c a l or pharmacological response i s observed and the plasma concentrat ion i s wel l above the expected therapeutic range i t i s u n l i k e l y that chronic therapy w i l l be e f f e c t i v e . F o l l a t h et a l . , (1983) have recent ly indica ted that knowledge of drug l e v e l s would improve the use of antiarrhythmic drugs i n the fo l lowing s i t u a t i o n s : (1) Lidocaine adminis t ra t ion to pat ients with congestive heart f a i l u r e and/or hypotension to suppress or prevent dangerous v e n t r i c u l a r arrhythmias af ter acute myocardial i n f a r c t i o n . In such cases l ignoca ine clearance i s often diminished but the c l i n i c a l f indings do not allow an est imation of the degree of pharmacokinetic a l t e r a t i o n s . Concomitantly administered vasoact ive drugs can also inf luence l ignoca ine requirements. With the recent ly introduced enzyme immunoassay, l i gnoca ine concentrations are measurable wi th in a few minutes and in fus ion rates can be adjusted according to i n d i v i d u a l needs. Routine determinations of l i d o c a i n e l eve l s would a lso increase the safety of high dose prophylac t ic l idoca ine adminis t ra t ion proposed by L i e et a l . , (1974) to prevent primary v e n t r i c u l a r f i b r i l l a t i o n . (2) Long term antiarrhythmic treatment i n pat ients with l i f e - t h r e a t e n i n g episodes of v e n t r i c u l a r tachycardia might be aided by plasma concentrat ion measurement i n two ways. F i r s t l y , the determination of - 4 -i n d i v i d u a l optimum concentrat ion range during the i n i t i a l phase of short term drug tes t ing can be e s t ab l i shed . In judging the adequacy of treatment, i t should be decided whether one intends to suppress a l l types of PrematureVentricular Contractions (PVCs) completely, or on ly episodes of v e n t r i c u l a r tachycard ia . Meyerberg et a l . (1981) have demonstrated that t o t a l PVC suppression requires higher plasma concentrat ion of the antiarrhythmic agent than e l imina t ion of sustained v e n t r i c u l a r tachycardia . Secondly, per iod ic measurements of plasma concentrat ions during follow-up provide an ob jec t ive c r i t e r i o n to assess pat ient adherence to the prescr ibed drug regimen and thus reduce the r i s k of treatment f a i l u r e by non-compliance. S i m i l a r l y , a l tered dose requirements by drug in te rac t ions or other diseases can also be detected i n th i s way. (3) Another obvious but less frequent i n d i c a t i o n for ant iarrhythmic drug l e v e l monitoring i s the necess i ty of dosage adaptat ion in pat ients with renal or hepatic f a i l u r e . B. Pharmacokinetics of Tocainide Tocainide (2-aminopropionq-2'6'=xylidide, f i g . 1) i s a r e l a t i v e l y new antiarrhythmic drug which i s s t r u c t u r a l l y re la ted to l idoca ine and e x h i b i t s s i m i l a r e l e c t r o - p h y s i o l o g i c a l , hemodynamic and antiarrhythmic e f f e c t s . In contrast to l i d o c a i n e , however, i t i s wel l absorbed af ter o r a l admin i s t r a t ion , has complete b i o a v a i l a b i l i t y and a plasma h a l f - l i f e of 11-13 hours i n healthy volunteers (Lalka et a l . , 1976; Graffner et a l . , 1980). In pat ients with myocardial i n f a r c t i o n , the mean h a l f - l i f e was also 13 hours, although the v a r i a b i l i t y was greater than that of -5-FIGURE 1 L I D O C A I N E - 6 -heal thy volunteers (Winkle et a l . , 1976; Ronfeld , 1980). Tocainide i s less potent than l idoca ine i n i t s d i r e c t cardiac e f f e c t s . Tocainide i s a primary amine i n contrast to the t e r t i a r y amine, l i d o c a i n e , and consequently the free base i s l e s s l i p o p h i l i c i n an octanol/water system (Ronfeld , 1980). Tocainide i s a bas ic drug (pka = 7.7) which i s spar ing ly soluble i n water. The more r e a d i l y soluble hydrochlor ide s a l t can be administered by the o r a l or intravenous route (Graffner et a l . , 1980; L a l k a , et a l . , 1976). It has been shown to be e f f ec t ive i n suppressing v e n t r i c u l a r arrhythmias i n both animals and man (Almotre f i and Baker, 1980; Schnittger et a l . , 1978; Moore et a l . , 1978; Duce et a l . , 1973; C o l t a r t et a l . , 1974; Young et a l . , 1980; Winkle et a l . , 1980; Roden et a l . , 1981; Sonnhag, 1980; Ryden et a l . , 1981; Waleffe et a l . , 1979; Koransky et a l . , 1981). In a number of t r i a l s with o r a l toca in ide given to pat ients with v e n t r i c u l a r ectopic beats (often fo l lowing a myocardial i n f a r c t i o n ) , doses of 1200 to 2400 mg d a i l y produced greater than 75% suppression of ec topic beats i n about 60% of the pat ients (Esterbrooks et a l . , 1983; Ryan et a l . , 1979). The chemical s tructure of toca inide contains an asymmetric center and the drug i s used c l i n i c a l l y i n the racemic form, containing equal quant i t i e s of each enantiomer. Pharmacological s tudies have shown that the R(-) enantiomer i s three times more potent than the S(+) isomer as an ant iarrhythmic agent i n a mouse model (Byrnes et a l . , 1979). The d i f ference i n the antiarrhythmic a c t i v i t y between the two enantiomers i s smaller i n coronary l i g a t e d dogs (Byrnes et a l . , 1979). - 7 -Tocainide i s ind ica ted for the treatment of v e n t r i c u l a r ectopic a c t i v i t y , using a dose of 400 mg every 8 h . 1. Absorption and Plasma Concentrations Fol lowing o r a l adminis t ra t ion of toca in ide hydrochlor ide to healthy vo lunteer s , absorption occurs r a p i d l y and b i o a v a i l a b i l i t y approaches 100%. The time to reach peak plasma l e v e l s a f ter a s ing le dose was approximately 1 h i n fas t ing subjects , while admini s t ra t ion with food decreased the peak l eve l s and delayed the rate of absorpt ion , but the b i o a v a i l a b i l i t y was not affected (Lalka et a l . , 1976). Several s tudies i n pat ients with myocardial i n f a r c t i o n who had received 400 mg of tocainide three times a day recorded mean plasma concentrat ions of between 5 and 7 ug/mL (Graffner et a l . , 1980). S imi lar l e v e l s were obtained i n patients with frequent v e n t r i c u l a r ec topic beats given 400 mg three times d a i l y (Meffin et a l . , 1977a; Winkle et a l . , 1976). When the dose was increased to 600 mg three times d a i l y i n these pa t i en t s , the mean plasma concentrat ions rose to about 9-10 ug/mL. 2. Intravenous Administration After intravenous i n f u s i o n , the plasma p r o f i l e of toca in ide can be described i n most cases by a b iexponent ia l equation (Graffner et a l . , 1980; La lka et a l . , 1976) with a rapid d i s t r i b u t i o n phase of about 10 minutes. Infusion of 300 mg over 30 minutes i n healthy subjects produced peak plasma l eve l s of approximately 2.5 Ug/mL (Lalka et a l . , - 8 -1976). In pat ients with myocardial ischaeraia, in fu s ion of 500 mg of toca in ide over 30 minutes resul ted i n plasma l eve l s of 11 ug/mL (Sutton, 1980). In a study of 7 pat ients with acute myocardial i n f a r c t i o n given a combination of 750 mg intravenous ly , d i r e c t l y followed by 800 mg o r a l l y , and subsequently 400 mg o r a l l y three times d a i l y , therapeutic plasma l e v e l s were atta ined i n most pat ients (4-10 ug/mL) wi th in 15 minutes and remained within the therapeutic range throughout the 48 hr period of observation (Graffner et a l . , 1980). S i m i l a r l y , when Baxter et a l . , (1982) administered 500 mg of toca in ide intravenously over 30 minutes to 14 pat ients with suspected myocardial i n f a r c t i o n , plasma l e v e l s ranged from 2.4 to 19.6 ug/mL fol lowing i n f u s i o n , with a mean value of around 5.8 ug/mL. 3. D i s t r i b u t i o n Toca in ide has been shown to enter the cerebrospinal f l u i d of dogs and sheep. Peak cerebrospinal f l u i d l e v e l s were achieved approximately 1 hour after intravenous in fu s ion of 5 mg/kg/min for 15 minutes. The r a t i o s between the plasma and cerebrospinal f l u i d l eve l s were found to be 1.2 to 1.6 and 1.1 to 1.5, 60 and 140 minutes, r e s p e c t i v e l y , a f ter the s tar t of the i n f u s i o n . Short ly a f ter intravenous and o r a l adminis t ra t ions of ( 3 H) toca inide i n mice, the drug was seen to concentrate i n the kidney, g a s t r i c mucosa, and the choroid plexus. Four hours l a t e r , high concentrations were found i n the wal l of the major a r t e r i e s . Nearly a l l of the r a d i o a c t i v i t y had disappeared a f ter 48 hours , apart from small amounts i n the kidney and the walls of the major a r t e r i e s (Holmes et a l . , 1983). - 9 -In man, a f ter in fus ion of 100 mg of toca in ide , the estimated h a l f - l i f e of the d i s t r i b u t i o n phase was 7 minutes i n healthy subjects and 13 minutes i n patients with acute myocardial i n f a r c t i o n (Graffner et a l . , 1980). The apparent volume of d i s t r i b u t i o n at steady state was found to be 1.46 L/kg on average i n healthy subjects r ece iv ing 300 mg intravenously (Lalka et a l . , 1976). Graffner et a l . (1980) reported values of 2.9 and 3.2 L / k g , r e s p e c t i v e l y , for the apparent volume of d i s t r i b u t i o n i n healthy subjects , given a 100 mg tocainide in fus ion and i n pat ients with acute myocardial i n f a r c t i o n , given infus ions of 750 mg of t oca in ide . E l v i n et a l . , (1982) reported a tocainide free f r a c t i o n of 0.78 to 0.96 i n sera from 10 normal volunteers and from 0.8 to 0.9 i n 4 trauma pat ients (who had high concentrat ions of <=^-acid glycoprotein) . Binding data, consis tent with the above study, were reported by Sedman et a l . (1982) using serum samples from f ive healthy volunteers . The percentage of to ta l unbound tocainide ranged from about 85 to 90%. Binding appeared to be independent of serum tocainide concentrations wi th in the range of 4-12 ug/mL. The p o s s i b i l i t y of a d i f ference i n binding of tocainide enantiomers has also been examined (Sedman et a l . , 1982). Although binding of the S(+) enantioraer was s l i g h t l y greater than that of R(-) isomer, the d i f ference would be of l i t t l e c l i n i c a l s i g n i f i c a n c e . 4. Metabolism and E l i m i n a t i o n E l v i n et a l . (1980b) studied the metabolism of tocainide in humans and reported a novel biotransformation pathway for a primary - 10 -amine, i n v o l v i n g the formation of the glucuronide of N-carboxy t o c a i n i d e . This g lucuronide , un l ike any previous ly reported, i s thought to undergo c y c l i z a t i o n to y i e l d a hydantoin-type compound. Approximately 30% of the dose has been recovered i n th i s form from u r i n e , together with 39 to 52% of the dose as unchanged drug, w i t h i n 72 h ( E l v i n et a l . , 1980a). Ronfeld et a l . , (1980) also i d e n t i f i e d a g lucuronide of l a c t o x y l i d i d e , an oxidat ive deamination product of t o c a i n i d e , which has a h a l f - l i f e of 26 h and therefore has the p o t e n t i a l to accumulate i n the body. The e l imina t ion of the l a c t o x y l i d i d e metabolite i s dependent on further metabolism. It seems u n l i k e l y that t h i s metabolite contr ibutes to the b i o l o g i c a l a c t i v i t y of the parent compound, as i t has been found to have n e g l i g i b l e pharmacological e f fec t i n animals (Ronfeld et a l . , 1980). The e l i m i n a t i o n h a l f - l i f e of toca inide i s 12 to 15 h . These values were obtained fo l lowing admini s t ra t ion by the intravenous and o r a l routes to healthy subjects and to var ious pat ient groups (Beat t ie et a l . , 1978; K l e i n et a l . , 1980; Meff in et a l . , 1977; Woosley et a l . , 1977; Graffner et a l . , 1980; La lka et a l . , 1976). Since hepatic degradation of toca inide i s a major route of e l i m i n a t i o n , the ef fects of hepatic enzyme inducers and i n h i b i t o r s on the rate of drug e l i m i n a t i o n i n both animals and humans have been i n v e s t i g a t e d . Several s tudies i n rats have demonstrated a decreased e l i m i n a t i o n h a l f - l i f e and area under the curve, as we l l as a lowered proport ion excreted as unchanged drug a f ter pretreatment with the enzyme-inducer, phenobarbital (Venkataramanan and Axelson, 1980a,b - 11 -Bennett et a l . , 1980, 1981). On the other hand, the enzyme i n h i b i t o r SKF-525A brought about an increase in e l imina t ion h a l f - l i f e and propor t ion of i n t a c t drug i n the ur ine and a decrease i n clearance (Venkataramanan and Axelson, 1980a,b). However, i n the only s i m i l a r study done i n man, there was no evidence of such a l t e r a t i o n s i n the d i s p o s i t i o n of toca in ide . Thus, E l v i n et a l . , (1980a) examined the e f f ec t of pre-treatment with phenobarbital and the competit ive substrates for the metabolis ing enzymes, sa l icy lamide and c l o f i b r a t e . I t was found that phenobarbital ( i n the therapeutic range of 15-40 ug/mL) did not induce any s i g n i f i c a n t change i n blood concentrations vs time p r o f i l e or the e l i m i n a t i o n rate constant. The proportions of tocainide to metabolites excreted i n urine were not a l t e r e d . S i m i l a r l y , the o v e r a l l pattern of recovery of tocainide and i t s glucuronide i n urine were not changed by sa l icy lamide or c l o f i b r a t e . 5. E f f e c t s of Disease on Pharmacokinetics Weigers et a l . , (1983) studied the d i s p o s i t i o n of tocainide i n 15 patients with renal dys funct ion . In 9 pat ients with end-stage rena l f a i l u r e ( c r e a t i n i n e clearance of less than 5 mL/min) the plasma h a l f - l i f e was s i g n i f i c a n t l y prolonged to about 27 h (range 16.6 to 42.7 h) and the to ta l plasma clearance ranged from 35 to 94 mL/min. The longest h a l f - l i f e (42.7 h) was found i n one pat ient with c i r r h o s i s i n a d d i t i o n to renal f a i l u r e . Three pat ients had h a l f - l i v e s longer than 30 h and a l l three were taking the enzyme i n h i b i t o r , a l l o p u r i n o l . In the remaining pat ients of this group, the h a l f - l i f e of tocainide ranged - 12 -from 16.6 to 27.5 h (22.3 ± 4.8 h) and the t o t a l plasma clearance from 52 to 94 mL/min (68 ± 18 mL/min). In s ix pat ients with impaired renal funct ion ( c rea t in ine c learance , 10-55 mL/min), the toca inide h a l f - l i f e ranged from 13.2 to 22 h (19.2 ± 4.0 h) and the t o t a l plasma clearance from 72 to 106 mL/min (90 ± 19 mL/min). During the 4 h of d i a l y s i s the h a l f - l i f e decreased to 8.5 ± 4.6 h which corresponds to removal of 25% of the drug from the body. Estimates of the apparent volume of d i s t r i b u t i o n were s i m i l a r to those reported i n healthy subjects (Lalka et a l . , 1976; Graffner et a l . , 1980). The d i s t r i b u t i o n volume of l i d o c a i n e , an analog of toca inide i s also unchanged i n pat ients wi th uremia (Thomson et a l . , 1973). From these f indings i t i s concluded that the e l i m i n a t i o n of toca inide i s impaired i n pat ients with renal dysfunct ion and that the dosage of tocainide must be adjusted for these p a t i e n t s . 6. Plasma Concentrations and C l i n i c a l E f f e c t s Studies r e l a t i n g toca inide plasma l eve l s to ant iarrhythmic a c t i v i t y vary i n t h e i r r e s u l t s , but genera l ly a therapeutic range of 4 to 10 ug/mL as the hydrochlor ide s a l t are considered acceptable (McDevitt et a l . , 1976; Winkle et a l . , 1976). V e n t r i c u l a r ectopic beats were suppressed by 20% i n pat ients taking tocainide (400 mg every 8 h) with plasma concentrations of 6 ug/mL and above, while only a s l i g h t increase i n suppression was seen i n pat ients given a higher dose of 600 mg every 8 h with plasma concentrat ion of 8.5 ug/mL. For the pat ients who responded to toca inide therapy, there was a c lear r e l a t i o n s h i p - 13 -between percentage reduct ion of v e n t r i c u l a r ectopic beats and plasma concentrat ion (Winkle et a l . , 1976; Meff in et a l . , 1977). Pat ients with s ide e f fects had serum concentrations between 7 and 16 pg/mL (Winkle et a l . , 1976). Winkle et a l . , (1978) also evaluated the long term e f f i c a c y of toca in ide i n 17 pat ients and obtained a s a t i s f ac to ry cont ro l of steady-state concentrations between 6.9 and 10.2 ug/mL. In a double b l i n d cross-over study i n 12 cases, Woosley et a l . , (1977) observed therapeut i ca l ly e f f ec t ive steady state toca in ide concentrat ions from 3.5 to 12 ug/mL. In more recent papers, Kuck et a l . , (1979) and Ryan and K a r l i n e r , (1979) reported e f f e c t i v e toca in ide plasma l e v e l s of 4.1 to 9.8 and 5-15 ug/mL r e s p e c t i v e l y . Toxic toca in ide concentrat ions i n Ryan's ser ies var ied between 7 and 18 ug/mL (mean 11.3 u g / m L ) . 7 . Side E f f e c t s The emergency use program i n the U . S . A . (Horn et a l . , 1980) made toca in ide ava i l ab l e for use i n the treatment of l i f e - t h r e a t e n i n g i n t r a c t a b l e v e n t r i c u l a r arrhythmias i n pat ients unresponsive to , or unable to take approved ant iarrhythmic drugs. This provided an opportunity to evaluate the safety of toca inide for periods of up to 41.5 months i n 369 s e r i o u s l y i l l pa t i en t s . The most commonly reported adverse e f fect s are nausea, vomit ing , anorexia , d i z z i n e s s , l ightheadedness , tremors, confusion, nervousness, p a l p i t a t i o n s , shortness of breath and rash. The majori ty of side ef fects are neuro log i ca l and g a s t r o i n t e s t i n a l i n nature. The neuro log i ca l - 14 -experiences appear to be re la ted to dosage, suggesting a poss ib le r e l a t i o n to peak blood l e v e l s . Adverse experiences causing terminat ion of therapy occurred i n 60 pat ients (16%). Neurologica l and g a s t r o i n t e s t i n a l ef fects were the primary causes of withdrawal (80% of withdrawals ) . Four pat ients discont inued toca inide because of the development of a rash , one because of h e p a t i t i s and one due to elevated l i v e r enzymes. Acute pulmonary edema developed i n one pat ient 5 h af ter drug admini s t ra t ion and another pat ient developed i n t e r s t i t i a l pulmonary edema. Two cases of poss ib le toca in ide associated h e p a t i t i s were reported by Lev in and Fox ,(1982). In a study of aggravation of v e n t r i c u l a r arrhythmias known to be caused by various ant iarrhythmic drugs, V a l e b i t et a l . , (1982) reported such an ef fect i n 12 out of 76 pat ients given 1200 to 2400 mg toca in ide for 48 h . Engler and LeWinter (1981) reported two cases of v e n t r i c u l a r f i b r i l l a t i o n induced by t o c a i n i d e . Both pat ients had severe underlying heart disease with l i f e - t h r e a t e n i n g recurrent v e n t r i c u l a r tachycardia but v e n t r i c u l a r f i b r i l l a t i o n d id not occur a f ter d i s cont inua t ion of therapy. Perlow et a l . , (1981) reported two cases of i n t e r s t i t i a l pneumonitis with c h a r a c t e r i s t i c c l i n i c a l r a d i o l o g i c a l and pulmonary funct ion changes af ter 6 months' use of 400 mg toca inide 4 times d a i l y . Withdrawal of toca in ide brought about improvement i n both cases. Brande et a l . , (1982) diagnosed a case of chronic d i f fuse i n t e r s t i t i a l pneumonitis i n a pat ient taking toca inide 400 mg 3 times d a i l y for 2 months. One week a f ter stopping toca inide and s t a r t i n g c o r t i c o s t e r o i d s the pat ient was much improved. Bradycardia has been reported i n a s soc ia t ion with - 15 -intravenous toca in ide i n 3 pat ients (Nyquist et a l . , 1980; Sutton, 1980). 8. Drug Interactions The e f fect of pretreatment with an enzyme inducer l i k e phenobarbital or co-admini s t ra t ion of a competit ive substrate for the metabol i s ing system such as sa l icy lamide or c l o f i b r a t e , has shown not to inf luence the e l imina t ion of tocainide i n humans ( E l v i n et a l . , 1980a). Metoprolol and toca in ide , s i n g l y or i n combination given to pat ients free from sinus node disease or impaired a t r i o v e n t r i c u l a r conduction had l i t t l e inf luence on e l e c t r o p h y s i o l o g i c a l or hemodynamic parameters. However, i n pat ients with ' S i c k - s i n u s ' syndrome and impaired a t r i o v e n t r i c u l a r conduction, the small depression of myocardial c o n t r a c t i l i t y may become s i g n i f i c a n t and should be avoided (Ikram, 1980; Renard et a l . , 1983). Concurrent admini s t ra t ion of d igoxin and toca inide produced no s i g n i f i c a n t d i f ference i n response to toca inide or a l t e red incidence of adverse e f fec t s compared with those pat ients given toca inide a lone. Tocainide did not af fect serum digoxin concentrations (Ryan et a l . , 1979). Rubino and Jackson (1982) reported a pat ient with severe paranoia fol lowing concomitant admini s t ra t ion of toca inide and p r o p r a n o l o l . Over a period of 16 months the pat ient was taking toca inide i n doses ranging from 600 to 2400 mg d a i l y . During th i s time he was treated with propranolol for unstable angina on two occas ions . Each time he suffered episodes of severe paranoia and confusion which subsided on withdrawl of p roprano lo l . - 16 -9. Comparison with Other Antiarrhythmic Drugs Toca in ide , given both intravenously and o r a l l y has been found to be at l e a s t as e f f ec t ive as procainamide i n suppression of v e n t r i c u l a r arrhythmias (Agnew and Whitelock, 1980; Sonnhag, 1980). In a report of a study on the comparative e f fects of tocainide and q u i n i d i n e , Wasenmiller and Aronow (1980) studying 22 pat ients with frequent v e n t r i c u l a r ectopic beats, found a greater response rate (based on 75% reduct ion i n v e n t r i c u l a r ectopic beats over placebo l eve l s ) i n the quin id ine treated pat ients (46% response rate) than i n the group treated with- tocainide (11% response r a t e ) . The comparative e f f i cacy of a lower dose of tocainide (600 mg twice da i ly ) against quinidine was studied by Morganroth et a l . , (1982). In this study tocainide was of comparable e f f i c acy to quinidine i n achieving greater than 75% reduct ion i n v e n t r i c u l a r ectopic beats. 10. Methods of Analysis of Tocainide i n B i o l o g i c a l F l u i d s A n a l y t i c a l methods for the determination of tocainide have included g a s - l i q u i d chromatographic and high-performance l i q u i d chromatographic techniques. The f i r s t GLC method reported consisted of conversion of tocainide to a tr i f luoroacetamide der iva t ive along with 1-aminoacetoxylidide as i n t e r n a l standard and analys i s of the r e s u l t i n g der iva t ive s on 5% OV-17 packed column (McDevitt et a l . , 1976). Both toca inide and the i n t e r n a l standard gave wel l - re so lved symmetrical peaks with r e t en t ion times of 4.0 and 5.9 minutes r e s p e c t i v e l y , and c a l i b r a t i o n curves for both plasma and urine were l i n e a r over the range - 17 -of 0.25 to 20.0 yg/mL. No i n t e r f e r i n g peaks were observed i n plasma or u r i n e . The method was su i tab le for measuring drug l eve l s up to 48 h . However, no s p e c i f i c data regarding chromatographic appearance or s e n s i t i v i t y and p r e c i s i o n of the method were inc luded . A GLC method (Venkataramanan and Axelson, 1978) using an e lec t ron capture detector ana lys i s of the hepta f luorobutyryl de r iva t ive was reportedly s e n s i t i v e down to 30 picograms of toca in ide . The a p p l i c a t i o n of th i s method to pharmacokinetic studies has been demonstrated by analys i s of tocainide i n 50-100 uL of r a t plasma (dose 20 mg/kg) c o l l e c t e d s e r i a l l y over a period of 3 h . Tocainide recovery from the plasma by the ex t rac t ion method used, s t a b i l i t y , as w e l l as s t ructure of the d e r i v a t i v e formed, have also been studied by these authors . La lka et a l . , (1976) and E l v i n et a l . , (1980b) employed a modified GLC method of C o l t a r t et a l . , (1974) which consisted of ana lys i s of the hepta f luorobutyryl d e r i v a t i v e (formed from tocainide and heptaf luoro-butyryl imidazole) on 3% OV-17 or 10% OV-17 packed column us ing e i ther a flame i o n i s a t i o n detector or e l e c t r o n capture detec tor . The monoheptafluorobutyryl d e r i v a t i v e of tocainide was stable only for 24 h ( E l v i n et a l . , 1980b). A gas chromatographic method based upon formation of a S c h i f f base with methylisobutylketone and employing a n i t rogen s e l e c t i v e detector has been reported by Johansson et a l . , (1982). The amine was extracted from a l k a l i n e samples with dichloromethane and, a f ter evapora-t i o n , the residue was reconst i tuted i n methylisobutylketone and heated i n a waterbath at 8 5 ° C for 10 mins. Af ter coo l ing to room temperature, - 18 -the s o l u t i o n was in jec ted d i r e c t l y in to the gas chromatograph using a carbowax 20 M (10%) packed column. The i n t e r n a l standard used was an analog of t oca in ide , containing an a d d i t i o n a l methyl group i n the p - p o s i t i o n of the benzene r i n g . The i n t e r n a l standard also formed a Sch i f f base with good chromatographic proper t i e s . The re ten t ion time of the toca in ide Schi f f base was 10 minutes and that of the i n t e r n a l standard, 13 minutes. The l i m i t of quant i t a t ion for th i s method was 96 ng/mL of plasma. High performance l i q u i d chromatography has also been employed for the determination of toca inide i n the plasma ( E l v i n et a l . , 1980b; Mef f in et a l . , 1977; Lagerstrom and Persson, 1978). Meff in et a l . , (1977) developed a very s e n s i t i v e HPLC method for measurement of toca in ide i n blood and plasma which involved the formation of a f luorescent d e r i v a t i v e with dansyl ch lor ide (5- (d imethylamino)- l -naphthalenesulphonyl ch lor ide ) and an i s o c r a t i c ana lys i s (mobile phase = 1:1 mixture of hexane and 2% methanol i n dichloromethane) using a u-Bondapack-amino column and f luorescent de tec t ion . The method has been shown to be useful for the measurement of therapeutic or subtherapeutic l e v e l s of the drug (0.1 to 5.0 ug/mL plasma) with a standard dev ia t ion of less than 2%. Although the s e n s i t i v i t y was adequate to measure therapeut ic plasma l e v e l s , the ex t rac t ion procedure was tedious and time consuming. Lagerstrom and Persson (1978) described an i o n - p a i r chromato-graphic technique ( p e r c h l o r i c a c i d , methanol, d ichloroethane , 0 .5 :10 :89 .5) using a P a r t i s i l column and UV de tec t ion . At a mobile - 19 -phase flow rate of 1.0 mL/min, the re tent ion time of tocainide was about 6 minutes. Another HPLC method ( E l v i n et a l . , 1980b) employed an oc tadecy l s i l ane (ODS) reverse-phase column and UV detec t ion . The mobile phase consisted of 25% a c e t o n i t r i l e i n 0.05 M NaClOit at pH 4 . The more recent HPLC method (Ronfeld et a l . , 1982), also an ion-pa i r technique, used a reverse-phase column, UV detect ion and a mobile phase c o n s i s t i n g of 0.01 M pentanesulfonic a c i d , 0.01 M dimethylhexylamine and 0.02 M KH2P0it i n methanol: a c e t o n i t r i l e : w a t e r , 15:15:70 with the pH adjusted to 3 .5 . This method was used for the measurement of two metabolites of toca in ide , the l a c t o x y l i d i d e formed by oxidat ive deamination, and toca inide carbamoyl glucuronide which was f i r s t converted to a hydantoin. Sedman et a l . , (1982) developed an HPLC method based on pre-column d e r i v a t i z a t i o n of tocainide with fluorescamine followed by f luorometr ic de tec t ion . The drug and chemical ly s i m i l a r i n t e r n a l standard, 2 -amino-6 ' -ch loro-o-ace to to lu id ide , were extracted from plasma with a c e t o n i t r i l e under sa l t ing-out condit ions obtained by sa turat ion of the aqueous medium with sodium chloride-sodium carbonate. The organic extract was treated with borate buffer (pH 8.2) and f luorescamine. The r e s u l t i n g der iva t ive s were chromatographed on an ODS reverse-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase. The authors also recommended that the d e r i v a t i z a t i o n reac t ion be ca r r i ed out immediately before HPLC a n a l y s i s . At room temperature, the fluorescamine i n t e n s i t y decreases s i g n i f i c a n t l y i n 1-2 h . However, the d e r i v a t i v e s can be stored for several hours i f cooled i n i c e . The product of the reac t ion of fluorescamine with racemic tocainide contains - 20 -two asymmetric centers and therefore, two diastereomeric der iva t ive s are formed. There was no evidence of a separation of diastereomers. C. Stereoselective Drug Analysis 1. Resolution of Diastereomers Drug enantiomers i n t e r a c t with c h i r a l molecules of l i v i n g organisms i n a s tereose lec t ive manner. As a r e s u l t , the pharmacological a c t i v i t y of enantiomers and the i r d i s p o s i t i o n (absorpt ion , d i s t r i b u t i o n , metabolism and excret ion) may d i f f e r . Methods for the analys i s of the i n d i v i d u a l enantiomers are therefore necessary i f s t e reose lec t ive d i s p o s i t i o n studies are to be ca r r i ed out. Since enantiomers d i f f e r only i n the ir c h i r a l propert ies ( o p t i c a l r o t a t i o n , i n t e r a c t i o n with other c h i r a l compounds) they are not reso lvable by the normal chromatographic techniques of drug a n a l y s i s . Reactions of enantiomers with an o p t i c a l l y pure d e r i v a t i z i n g reagent leads to formation of diastereomers. These have d i f f e r e n t chemical and phys ica l propert ies and are thus, i n p r i n c i p l e , separable by chromatographic means (HPLC, GLC, TLC) . Appropr ia te ly chosen d e r i v a t i z i n g reagents can f a c i l i t a t e the chromatographic process as wel l as d i f f e r e n t i a t e between enantiomers. Disadvantages associated with c h i r a l d e r i v a t i z a t i o n reagents include the requirement of an act ive funct iona l group for the formation of d ias tereoisomeric d e r i v a t i v e s , d i f ferences i n the r eac t ion rates of d i f f e r e n t pairs of enantiomeric compounds and d i f f i c u l t i e s i n obta in ing o p t i c a l l y pure reagents (Konig et a l . , 1977). In a d d i t i o n , - 21 -the method cannot be employed i f the diastereomerlc mixture i s not chemical ly and stereochemical ly stable under chromatographic c o n d i t i o n s . Although c a p i l l a r y columns were f i r s t used i n gas chromato-graphic analys i s (Raban and Mislow, 1967), newer o p t i c a l l y act ive reagents permit the use of packed columns. The most commonly used reagent has been N - t r i f l u o r o a c e t y l -S - p r o l y l c h l o r i d e (TPC) (Wel l s , 1970; Westley et a l . , 1968; Gord i s , 1966; Dabrowiak and Cook, 1971). Other reagents include S(-)-N-pentaf luorobenzoyl p r o l y l - l - i m i d a z o l i d i d e (PFBP) (Chat ter j i e et a l . , 1974), and S(-) ot-methoxy-ot-trifluoromethylphenylacetylchloride ((S)-MTPA) (Gal et a l . , 1982; G a l , 1977; N i c h o l s , et a l . , 1973). These reagents are acy l a t ing agents and d i scr iminate between enantiomeric amines and a l coho l s . Gal (1977) used the l a t t e r reagent to resolve amphetamine and e ight re la ted compounds. 2. D i r e c t Resolution of Enantiomers With the recent i n t r o d u c t i o n of c h i r a l s ta t ionary phases, the d i r e c t GLC r e s o l u t i o n of enantiomers i s po s s ib l e . G i l - A v et a l . , (1966, 1967) f i r s t introduced the use of N - t r i f l u o r o a c e t y l (N-TFA) L - i s o l e u c i n e l a u r y l ester for the separation of N-TFA ot-amino acid esters on w a l l -coated c a p i l l a r i e s . The operating temperature of th i s phase i s 9 0 ° C and therefore only h ighly v o l a t i l e der iva t ive s of amino acids can be analysed. Recognizing the e s s e n t i a l ro le of the -NHCO*CH(R)NH.CO-group i n these type of peptide ester phases, the search for ce r t a in s t r u c t u r a l features of amides which might increase s e l e c t i v i t y as wel l as thermal - 22 -s t a b i l i t y led to the development of N - l a u r o y l - L - v a l y l - t e r t . b u t y l a m i d e phase (Feibush, 1971). Subsequent diamide phases derived from L - v a l i n e exhib i ted greater e f f i c i e n c y , higher r e s o l u t i o n factors and reduced re tent ion times ( B e i t l e r and Feibush, 1976). To obtain bet ter thermal s t a b i l i t y and lower v o l a t i l i t y , L - v a l i n e - t e r t . b u t y l a m i d e was coupled to the carboxyl group of the co-polymer of dimethyls i loxane and carboxyalkylmethyls i loxane (Frank et a l . , 1977). This phase was wal l coated on c a p i l l a r i e s and i s designated as C h i r a s i l - V a l ® and has been used to separate enantiomeric drugs and metabolites (Frank et a l . , 1978; L i u , et a l . , 1982), amino acids of high and low v o l a t i l i t y and some amino a lcohols (Solomon and Wright, 1977, Frank et a l . , 1979; Frank et a l . , 1980). In a d d i t i o n , the high thermal s t a b i l i t y of C h i r a s i l - V a l ® made i t poss ib le for the f i r s t time to employ a mass spectrometer coupled to GLC system for the analys i s of enantiomers ( L i u et a l . , 1981; Frank, et a l . , 1978). L - v a l i n e - t e r t . b u t y l a m i d e has also been incorporated into a wel l known GLC s ta t ionary phase, polycyanopropyl phenylmethyls i l i cone (0V-225). The r e s o l u t i o n of norephedrine enantiomers was accomplished on th i s phase (Saeed et a l . , 1979). 3. Resolution of Enantiomers by HPLC Like GLC methods, HPLC has also been used to resolve enantiomers. Most of the e a r l i e r methods reported have dealt with separat ion of amino acids using a v a r i e t y of d e r i v a t i z i n g reagents (Baczuk, et a l . , 1971; Lefebvre et a l . , 1978). More r e c e n t l y , i o n - p a i r chromatography has been used to resolve enantiomeric amines (Pettersson - 23 -and S c h i l l , 1981). This method i s based on ion-pa i r chromatography with a c h i r a l counter- ion added to the mobile phase. Using (+) camphor sulphonic acid as the counter - ion , these authors were able to resolve enantiomers of a l p r e n o l o l , metoprolol and proprano lo l . Propranolol was a lso resolved into i t s enantiomers by formation of a diastereomeric d e r i v a t i v e with N - t r i f l u o r o a c e t y l - s - p r o l y l c h l o r i d e followed by chromatography on a reverse-phase column with f luorometric detec t ion (Hermansson and Bahr, 1980). A s i m i l a r approach has been used to reso lve enantiomers of warfarin i n the plasma (Banf ie ld and Rowland, 1983). This method involved the formation of d ia s ter iomer ic e s t e r s , using carbobenzyloxy-L-prol ine , with subsequent separation using s i l i c a as the s ta t ionary phase. The method permits c h a r a c t e r i z a t i o n of the pharmacokinetics of warfarin enantiomers fol lowing admini s t ra t ion of the racemic drug. C h i r a l s ta t ionary phases also have been developed for r e s o l u t i o n of enantiomers by HPLC. P i r k l e et a l . , (1980) developed c h i r a l s t a t ionary phases such as 3 ,5 -d in i t robenzoy l phenylglycine attached to a m i n o p r o p y l - s i l i c a ge l for l i q u i d chromatographic separation of enantiomeric amines and a l c o h o l s . This column i s now commercially ava i l ab le i n e i ther the i o n i c a l l y or cova len t ly bonded form. 4. Ste r e o s p e c i f i c Radioimmunoassays Since antibodies are c h i r a l compounds, the complexes formed when they bind to enantiomers exh ib i t d i f f e r i n g s t a b i l i t y constants . Thus, i t i s poss ib le to prepare ant i se ra to i n d i v i d u a l enantiomers of a drug - 24 -and use the ant i se ra for enantiomer a n a l y s i s . In genera l , exce l lent s e l e c t i v i t y can be achieved for one enantiomer i n the presence of the other . Recent ly , Midha et a l . , (1983) developed a s t e r e o s p e c i f i c radioimmunoassay for d- and 1-ephedrine i n human plasma and have used the technique to fol low the plasma concentration.-:time curve af ter an o r a l dose of the racemate. 5. Resolution of Tocainide Enantiomers A g a s - l i q u i d chromatographic method based on conversion of the c h i r a l amine to a diastereomer has been reported by G a l , et a l . , (1982). The c h i r a l d e r i v a t i s i n g reagent used was (S)-ct-methoxy-ct-t r i f l u o r o p h e n y l a c e t y l ch lor ide (MTPA). When toca inide was mixed with th i s reagent and heated at 60 - 6 5 ° C for 30 minutes the diastereomer was formed. Pyr id ine was used as a ca ta ly s t i n th i s a c y l a t i o n r e a c t i o n . The d e r i v a t i v e had to be i s o l a t e d by an acid-wash with hydroch lor i c ac id (1.0 N) followed by washing with sodium carbonate so lu t ion (15%). For gas chromatographic analys i s of the d e r i v a t i v e s , a conventional packed column (3% OV-17) and a nitrogen-phosphorus detector was used. The lower l i m i t of s e n s i t i v i t y of the assay was 1.0 ug/mL for each enantiomer and therefore a r e l a t i v e l y large sample s ize (1 mL serum or 1 mL urine) had to be used. The re tent ion times of the MTPA d e r i v a t i v e of S(+) toca in ide was 13 mins and that of R(-) t o c a i n i d e , 11.3 mins. D i rec t Reso lut ion of Tocainide Enantiomers Recently McErlane and P i l l a i (1983) developed a g a s - l i q u i d chromatographic method for the d i rec t r e s o l u t i o n of toca in ide - 25 -enantiomers employing a f u s e d - s i l i c a c a p i l l a r y column coated with an o p t i c a l l y ac t ive s ta t ionary phase, N - i s o b u t y r y l - L - v a l i n e - t e r t . -butylamide. C a p i l l a r y columns coated with th i s s ta t ionary phase are commercially ava i l ab le as C h i r a s i l - V a l ® and have been demonstrated to be e f f e c t i v e for r e so lu t ion of amino acids and amino a lcohols (Frank, et a l . , 1980). This phase offers exce l lent r e so lu t ion of the enantiomers of toca inide as t h e i r heptaf luorobutyrate d e r i v a t i v e s . McErlane and P i l l a i (1983) and P i l l a i et a l . (1984) used th i s technique for the simultaneous measurement of toca in ide enantiomers i n human plasma, urine and s a l i v a fo l lowing o r a l or intravenous admini s t ra t ion of the racemate. The lower l i m i t of s e n s i t i v i t y of the assay was 25 ng/mL (e l ec t ron capture detector) for each enantiomer using the split-mode of i n j e c t i o n . D. Renal F a i l u r e and Drug Accumulation In view of the extent of ur inary excret ion of toca inide (40% of dose as i n t a c t drug i n normal subjects and (20-70%) i n disease states) and i t s major metabolite TOCG (25-40% dose), the ef fect of renal dysfunct ion on the e l imina t ion of these substances should be quanti tated to permit r a t i o n a l dosing adjustment i n rena l disease and during d i a l y s i s . The time period during which a drug stays i n the body and also the i n t e n s i t y and durat ion of pharmacological ef fects are functions of the rate and extent of drug absorpt ion, d i s t r i b u t i o n , metabolism and e l i m i n a t i o n . Some drugs are c leared from the body predominantly i n t h e i r unchanged form while others are p a r t i a l l y or completely - 26 -metabolised and then cleared from the body predominantly by the k idneys . Thus, whether a drug i s c leared from the body i n unchanged form, or extens ively metabolised, i t s o v e r a l l pharmacologic p r o f i l e i s p a r t i a l l y a funct ion of i n t e g r i t y of renal func t ion . When rena l funct ion i s compromised through rena l disease, drugs el iminated predominantly by the kidneys w i l l tend to be reta ined i n the body and may accumulate to tox ic l eve l s with repeated dos ing . If a drug i s converted to a metabol i te , then accumulation of the metabolite may also lead to tox ic e f f ec t s . The prolonged and progressive nature of chronic renal f a i l u r e i s of p a r t i c u l a r concern i n older pat ients who may require a v a r i e t y of medicat ions, both for t h e i r basic rena l condi t ion and for re la ted or unrelated f a c t o r s . The i n a b i l i t y of these pat ients to excrete drugs adequately requires care fu l dosage adjustment to obtain adequate therapeutic blood l eve l s without increased t o x i c i t y . Procainamide and i t s ac t ive N-acetyl metabolite have been shown to accumulate i n renal dysfunction (Drayer et a l . , 1977). Renal f a i l u r e caused large reductions (2-4 fo ld) i n t o t a l body clearance (with concomitant increase i n t\/2) of metoclopramide i n man (Bateman and Gokhal , 1980) and rat (Tam et a l . , 1981), despite the fact that both species only excrete 20% of the dose as in tac t drug. In rena l i n s u f f i c i e n c y (glomerular f i l t r a t i o n rate < 5 mL/min) the h a l f - l i f e of a m p i c i l l i n i s increased from 1 h to 10-18 h (Kunin, 1970); t e t r a c y c l i n e from 8.5 to 57-108 h (Kunin et a l . , 1959); gentamicin from 2.3 to 35-67 h (McHenry et a l . , 1971); vancomycin from 6 to 200 h (Kunin, 1967; Lindholm and Murray, 1966) and chlorpropamide from 36 to 200 h ( P e t i t p i e r r e et a l . , 1972). - 27 -In add i t ion to the parent drug, drug metabolites which may be pharmacological ly a c t i v e , can also accumulate i n renal i n s u f f i c i e n c y i f r ena l excret ion i s a major pathway of e l i m i n a t i o n . The high incidence of adverse drug react ions seen i n pat ients with renal f a i l u r e may, for some drugs, be p a r t l y explained by accumulation of ac t ive drug metabol i te s . Norpethidine i s a metaboli te of pethidine with le s s analges ic but more convulsant a c t i v i t y (Denean and Nakai , 1961). This metaboli te accumulates slowly with mul t ip l e doses of pethidine i n most cancer pa t i en t s , but r a p i d l y i n pat ients with renal f a i l u r e . Severe i r r i t a b i l i t y and twitching has been noted i n uremic pat ients who have been treated with pethidine and these are associated with high l e v e l s of norpethidine (Szeto et a l . , 1976). The severe muscle weakness and tenderness seen i n pat ients with rena l f a i l u r e r ece iv ing c l o f i b r a t e are associated with excessive accumulation of the free acid metabol i te , chlorophenoxyisobutyric acid (P ie r ides et a l . , 1975). Accumulation of free and acetylated sulphonamides i n pat ients with renal f a i l u r e i s associated with an increase i n tox ic side ef fects (severe nausea and vomit ing , Weinste in, 1975). In add i t ion to the r i s k of tox ic •accumulat ion of drugs and/or t h e i r metabolites i n renal i n s u f f i c i e n c y , c e r t a i n p h y s i o l o g i c a l and anatomical changes also a l t e r the pharmacokinetic parameters of drugs, making dosage modi f i ca t ion even more d i f f i c u l t . The volume of d i s t r i b u t i o n of drugs such as d igoxin (Reuning et a l . , 1973), cephalex in , m e t h i c i l l i n and l incomycin ( G i b a l d i and P e r r i e r , 1972) have been reported to decrease due to changes i n t h e i r d i s t r i b u t i o n c h a r a c t e r i s t i c s i n renal impairment. - 28 -Reduction i n renal blood flow and glomerular f i l t r a t i o n rate are associated with re tent ion of sodium and water. These events can lead to an increase i n the volume of d i s t r i b u t i o n of c e r t a i n drugs and decrease i n steady state plasma concentra t ion . For ins tance , L e t t e r ! et a l . , (1971) found that fo l lowing bolus i n j e c t i o n s of phenytoin, plasma concentrat ions of th i s drug were lower i n pat ients with impaired renal funct ion than i n normal sub ject s . S i m i l a r l y Odar-Cederlof and Borga (1974) and Odar-Cederlof (1977) reported that the plasma concentrat ions of phenytoin and warfarin were lower and the volume of d i s t r i b u t i o n was higher i n uremic pat ients than i n pat ients without renal d i sease . Funct iona l and anatomical changes to the g a s t r o i n t e s t i n a l t rac t caused by chronic uremia may af fect to some degree the absorption of drugs given o r a l l y . Serum concentrations of chloropropamide ( P e t i t p i e r r e et a l . , 1972) and doxycycl ine (Fabre et a l . , 1967) are lower i n rena l pa t ients than normal subjects a f ter r ece iv ing s i m i l a r o r a l doses. The inf luence of kidney disease on drug metabolism has been reviewed (Reidenberg, 1971; Reidenberg, 1974; Reidenberg, et a l . , 1978). Recent studies have led to the observation that drug oxidat ions occurr ing i n the endoplasmic ret iculum of the l i v e r are accelerated i n uremia. The f i r s t observation of th i s sort was that of L e t t e r i et a l . (1971) who observed more rapid e l i m i n a t i o n of phenytoin i n pat ients with renal f a i l u r e than i n normal sub jec t s . This was confirmed by Odar-Cederlof (1974) who proved that the rapid e l imina t ion of phenytoin i n uremic pat ients was due to rapid metabolic c learance . Another drug ox id i sed by the l i v e r that has been found to have accelerated metabolism - 29 -i n pat ients with rena l f a i l u r e i s a n t i p y r i n e . An increased ant ipyr ine e l i m i n a t i o n rate has been observed i n both d ia lysed and undialysed pat ients with chronic renal f a i l u r e ( L i c h t e r et a l . , 1973; Maddocks et a l . , 1975). E. Glucuronldation i n Renal F a i l u r e Glucuronides are a c i d i c compounds (pka 3.5-4) which are f u l l y ioni sed i n plasma and u r i n e , and are r e a d i l y el iminated from the body by the kidneys . Glucuronides of some drugs are also excreted to a s i g n i f i -cant extent into the b i l e , with molecular weight, chemical s tructure and p o l a r i t y of the glucuronide determining the extent of b i l i a r y excret ion (Levine , 1978; R o l l i n s and Klas sen , 1979). Although excret ion of glucuronides in to the urine r e s u l t s i n the i r r e v e r s i b l e e l i m i n a t i o n of the conjugate from the body, this i s not nece s sa r i ly true i f the glucuronide i s excreted in to the b i l e . Fol lowing b i l i a r y excre t ion , glucuronides may become substrates for the m i c r o b i a l glucuronidase of the g a s t r o i n t e s t i n a l t r ac t , permit t ing reabsorption of the l i b e r a t e d parent compound (Okolicsanye et a l . , 1971; H a r t i a l a , 1973). Hydro lys i s of glucuronides can also be ca r r i ed out by lysozomal ^-glucuronidase which i s present i n most t i s sues , p a r t i c u l a r l y l i v e r , kidney, spleen, i n t e s t i n a l epi thel ium and endocrine and reproductive organs (Levy and Couchie, 1966; Wakabayashi, 1970). Glucuronide conjugation has not been ex tens ive ly inves t iga ted i n pat ients with renal f a i l u r e . Studies i n r a t s , however, have shown that the g lucuronidat ion process may be diminished (phenolic a c i d , p-aminobenzoic a c i d ) , increased (naphthol) - 30 -or unaffected (4-methylumbelliferone) i n experimental rena l f a i l u r e (Canalese et a l . , 1980; Howie and Bourke, 1979; Leber et a l . , 1972). Despite the s c a r c i t y i n experimental data i n pat ients with rena l f a i l u r e , the g lucuronidat ion process i s genera l ly categorized as unaffected by th i s disease state (Reidenberg, 1978). However, recent studies have shown that f a i l u r e to excrete a glucuronide may lead to h y d r o l y s i s of the conjugate, leading to accumulation of the ac t ive compound (Verbeeck, 1982). The a n t i l i p e m i c agent, c l o f i b r a t e i s biotransformed to an ester-type glucuronide (60% of dose) (Gugler et a l . , 1979b). Despite t h i s , the plasma e l i m i n a t i o n h a l f - l i f e of chlorophenoxyisobutyric acid (CPIB), the act ive form of c l o f i b r a t e , was 2-6 f o l d longer i n rena l f a i l u r e pat ients (Faed et a l . , 1979; Gugler , 1979a). S i m i l a r l y , i n a recent , s ingle-dose study, i t was shown that the t o t a l body clearance of d i f l u n i s a l , a recent ly developed s a l i c y l a t e ana lges ic , was s i g n i f i c a n t l y decreased i n pat ients with rena l f a i l u r e (De Schepper et a l . , 1977; Verbeeck et a l . , 1979). D i f l u n i s a l also forms an es ter- type glucuronide (Tocco et a l . , 1975). Poss ib le mechanisms include a l t e red g lucuronidat ion of d i f l u n i s a l i n renal f a i l u r e , b i l i a r y excre t ion of one or both of the glucuronides followed by hydro lys i s i n the i n t e s t i n a l t rac t and reabsorption of unchanged d i f l u n i s a l , and systemic deconjugation of one or both of the g lucuronides , as has been described for the ester-type of glucuronide of CPIB (Faed, 1980; Levy, 1979; De Schepper et a l . , 1977). Glucuronide accumulation i n renal f a i l u r e pat ients has also been shown for the benzodiazepines, lorazepam (Verbeeck et a l . , 1976) and oxazepam (Odar-Cederlof et a l . , 1977). - 31 -F. E n a n t i o s e l e c t l v i t y i n Drug D i s p o s i t i o n O p t i c a l isomers of many drugs have been shown to d i f f e r i n t h e i r pharmacological a c t i v i t i e s due to t h e i r a b i l i t y to react s e l e c t i v e l y with an asymmetric center i n the b i o l o g i c a l system (Casy, 1970; P a t i l et a l . , 1975; Ramasastry, 1973). The observed di f ferences i n b i o l o g i c a l a c t i v i t y may be due to a d i f ference i n the propert ies of the drug-receptor combination. Dif ferences i n d i s t r i b u t i o n occur as o p t i c a l isomers are se lected by some other asymmetric center i n the b i o l o g i c a l system before the isomer reaches the s p e c i f i c receptor . This may be due to o p t i c a l l y ac t ive processes such as s e l ec t ive penetrat ion of membranes during transport ( P a t i l et a l . , 1975; Ames et a l . , 1977; Gray, 1976), or s e l e c t i v e metabolism (Jenner and Tes ta , 1980; Low and Cas tagno l i , 1978). An o p t i c a l l y ac t ive drug may not be subjected to a l l of these processes but they may contr ibute to s u p e r i o r i t y of b i o l o g i c a l e f fect of one isomer. 1. Enantioselective D i s t r i b u t i o n The two main factors in f luenc ing the plasma h a l f - l i v e s of phenprocoumon and warfar in enantiomers are t h e i r b inding to serum prote ins and t h e i r biotransformation (Breckenridge and Orme, 1972; Schmidt and Jahnchen, 1977; Jahnchen et a l . , 1976). The uptake of drugs by var ious organs may be enant io se lec t ive as shown by phenprocoumon. (Schmidt and Jahnchen, 1977) where the more potent enantiomer i s s e l e c t i v e l y taken up by the l i v e r . Another example of s t e reose lec t ive uptake i s seen with the ant imuscar in ic drug benzetinide, the - 32 -pharmacological ly potent S(+) enantiomer of which i s s e l e c t i v e l y taken up in to guinea pig cardiac t i s s u e s . The cardiac accumulation was found to be almost e n t i r e l y due to non- spec i f i c b ind ing . The non-spec i f i c binding s i t e s therefore d i sp lay a c e r t a i n s t e r e o s e l e c t i v i t y but to a much smaller extent than the pharmacological receptor binding s i t e s (Gray et a l . , 1976). Another example i s provided by the B-adrenergic b locking agent, p roprano lo l . Fol lowing intravenous admini s t ra t ion of the racemate to r a t s , the serum and heart concentrations of the i n d i v i d u a l enantiomers were monitored. Af ter 5 minutes, the serum l e v e l of the S(-) form was three times lower than that of i t s antipode. During the fo l lowing 4 h , the (+) form disappeared much fas ter from the serum than the (-) form. The i n i t i a l l y lower l e v e l of the (-) form i s explained by a h igh ly s e l e c t i v e cardiac uptake. Most of the propranolo l found i n the heart was the (-) isomer. It thus appears that the ac t ive enantiomer i s s e l e c t i v e l y accumulated i n i t s organ of ac t ion whereas the (+) isomer remains i n the blood and i s r a p i d l y metabolised (Kawashima et a l . , 1976). S tereose lec t ive uptake of propranolol has a lso been found i n the heart and bra in of mice (Levy et a l . , 1976). An i n t e r e s t i n g case of s t e reose lec t ive l i v e r uptake and enantiomeric i n t e r a c t i o n has been documented for propoxyphene. Only the (+) isomer shows analges ic a c t i v i t y i n the r a t . A 20 mg/kg dose of th i s isomer i n the rat i s equ i -ac t ive with a combination of two enantiomers at a dose of 10 mg/kg each. The explanat ion for th i s s y n e r g i s t i c e f fect was found i n the d i s p o s i t i o n of the two enantiomers. When dextro-propoxyphene was infused i n the i s o l a t e d perfused rat l i v e r , over 98% of the drug was - 33 -extracted i n a s ing le pass. When the levo-isomer was added to the perfusate at the same concentra t ion , the ex t rac t ion of the dextro-isomer was reduced to less than 90%. A s i m i l a r s i t u a t i o n preva i l s i n v ivo where the plasma l e v e l s of dextro-propoxyphene increased s e v e r a l - f o l d upon simultaneous dosing of i t s enantiomer. It therefore appears that the levo-propoxyphene must be p r e f e r e n t i a l l y bound to uptake or metabolic s i t e s wi th in the l i v e r , r e s u l t i n g in the release of dextro-propoxyphene into the systemic c i r c u l a t i o n and hence i t s increased systemic a v a i l a b i l i t y (Murphy et a l . , 1976). 2. Enantioselective Metabolism In most ins tances , s t e reose lec t ive drug d i s t r i b u t i o n may occur concomitantly with s te reose lec t ive metabolism and the l a t t e r often determines the o v e r a l l d i s p o s i t i o n of the drug. Dif ferences i n the d i s p o s i t i o n of propranolol enantiomers have also been reported (Kawashima et a l . , 1976; George et a l . , 1972; Ehrson, 1975). Slower metabolism, longer h a l f - l i f e , more 0-glucuronide formation and more s t e reo se l ec t ive uptake have been noted for the S(-) isomer. Numerous metabolites of propranolol have been character ized but enant io se lec t ive d i f ferences have been reported only for the O-glucuronide (Ehrson, 1975). Enant io se lec t ive metabolism of the antiarrhythmic agent, drobul ine has been reported (Murphy et a l . , 1977). Three- fo ld higher plasma l e v e l s of the (-) isomer were observed when dogs were dosed with the racemate or with i n d i v i d u a l enantiomers. More rapid metabolism of the (+) isomer appears to account for the d i f ferences i n plasma l eve l s - 34 -of the two enantiomers. C h i r a l l o c a l anesthetic agents have also been demonstrated to exh ib i t enant iose lec t ive d i s p o s i t i o n (De Jong 1977). The less act ive R(-) isomer of p r i l o c a i n e i s p r e f e r e n t i a l l y hydrolyzed and may be responsible for i t s greater methemoglobinemic t o x i c i t y . Absorpt ion of the act ive S(+) enantiomer of both mepivacaine and bupivacaine i s more rapid than that observed for the more toxic R(-) ant ipodes . Numerous other examples of enant iose lec t ive metabolism have been reported by Jenner and Testa (1980). 3. Inversion of Configuration as a Metabolic Process Conf igura t iona l inver s ion i s genera l ly considered as invo lv ing con f i gura t iona l s t a b i l i t y of one isomer while the other isomer undergoes slow or rapid conversion to the former. Stereochemical inver s ion was f i r s t postulated for the anti-inflammatory drug, ibuprofen. Fol lowing admini s t ra t ion of racemic ibuprofen or i t s i n d i v i d u a l isomers to several spec ies , the major ur inary metabolites were shown to be dextro-rotatory ( M i l l s et a l . , 1973). The unchanged drug excreted was also found to be enriched i n the dextro-rotary isomer (Brooks and G i l b e r t , 1974; Vangiessen and K a i s e r , 1975). There appears to be almost complete i n v e r s i o n of the much less ac t ive (-) form to the much more act ive (+) enantiomer i n the mouse, rat and guinea pig (Adams et a l . , 1976). The stereochemical composition of the products excreted i n human urine a f ter admin i s t ra t ion of ibuprofen, reveals a s i g n i f i c a n t but incomplete conversion of (-) enantiomer to the (+) form (Kaiser et a l . , 1976). The process of conf igura t iona l inver s ion as exemplif ied by ibuprofen i s - 35 -common to a number of anti-inflammatory 2-ary lprop ionic a c i d s . C ic lopro fen undergoes (-) to (+) conversion i n the dog, r a t , monkey and man ( K r i p a l a n i et a l . , 1976; Lan et a l . , 1976). It i s also shown recent ly that R(-) benoxaprofen i s s t e r e o c h e m i c a l ^ inverted to the S(+) enantiomer by rats and humans. The rate of inver s ion i s much fas ter i n the rats than i n human (Simmonds et a l . , 1980). These authors also suggested that th i s transformation does not occur i n the l i v e r but occurs while passing through the gut w a l l s . If inver s ion does occur i n the gut w a l l , the extent of inver s ion of R(-) benoxaprofen i n various species could be re la ted to the degree of enterohepatic c i r c u l a t i o n . Thus r a t s , which excrete benoxaprofen p r i m a r i l y i n the feces v i a the b i l e (Cha t f i e ld and Green, 1978) and have the p o t e n t i a l to reabsorb the compound, are rapid i n v e r t e r s . Whereas humans, who excrete a high proport ion of the dose i n the urine (Smith et a l . , 1977) with correspondingly less b i l i a r y exc re t ion , are slow inver ter s of the compound. G. S p e c i f i c Alms of the Project 1. The chemical s t ructure of toca in ide contains an asymmetric center and therefore the molecule can ex i s t i n two isomeric forms. The racemic mixture , which contains equal proportions of the dextro- and l evo- ro ta to ry isomers, i s used t h e r a p e u t i c a l l y as an ant iarrhythmic agent. Although the antiarrhythmic potency of toca inide enantiomers has not been studied i n man, the R(-)enantiomer i s three- fo ld more potent than the S(+)isomer as an ant iarrhythmic agent i n a mouse model. - 36 -Differences i n ant iarrhythmic a c t i v i t y between the enantiomers have a lso been demonstrated i n coronary- l iga ted dogs. In a d d i t i o n , d i f ferences i n plasma l e v e l s of the enantiomers as w e l l as ur inary excret ion have been observed i n mice and r a t s . A packed-column gas chromatographic method employing diastereoisomer formation and n i t r o g e n - s e l e c t i v e detec t ion was used i n these s tud ie s . The lower l i m i t of s e n s i t i v i t y of the assay method was 1 mg/mL plasma for each enantiomer and therefore would not be su i t ab le for the c h a r a c t e r i s a t i o n of pharmacokinetic parameters of toca in ide enantiomers i n human, e s p e c i a l l y when low doses are administered. This has necess i tated the development of a s t e reo se l ec t ive assay method that not only would resolve the two isomers but a lso would have high s e n s i t i v i t y to measure the low nanogram l e v e l s of i n d i v i d u a l isomers i n b i o l o g i c a l f l u i d s . Since c a p i l l a r y column gas chromatographic methods are superior to conventional gas chromatographic procedures i n terms of r e s o l u t i o n c a p a b i l i t y and s e n s i t i v i t y of d e t e c t i o n , the aim was to develop a c a p i l l a r y gas chromatographic method. Moreover, 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 c a p i l l a r y columns combined with e l ec t ron capture detect ion would be most su i t ab le for enantiomer d i s p o s i t i o n studies s ince th i s would obviate the need for formation of diastereomers with o p t i c a l l y pure reagents. 2. Since toca inide had never been assayed by a c a p i l l a r y column gas chromatographic method u n t i l th i s study was i n i t i a t e d , i t was necessary to study the f e a s i b i l i t y of the technique by analyzing extremely low l e v e l s of the drug i n plasma and urine and comparing the re su l t s obtained with published data . It was also necessary to - 37 -v a l i d a t e the s te reose lec t ive assay by an independent determination of the racemate. This has necess i tated the development of a non-s tereose lec t ive c a p i l l a r y column gas chromatographic method. It was decided to use small laboratory animals such as rats during the i n i t i a l developmental phase i n order to compare ear ly pharmacokinetic measurements with previous ly published repor t s . 3. Tocainide i s reported to be metabolised by a novel pathway to a glucuronide of N-carboxytocainide. The assignment of the s tructure of th i s metabolite was based on i n d i r e c t evidence such as conversion to 3- (2 ,6-xy ly l ) -5-methyl hydantoin. This hydantoin d e r i v a t i v e can be formed not only from the glucuronide of N-carboxy toca in ide but also by reac t ion between phosgene and tocainide as shown by Johansson et a l . , (1982). Therefore i t was of i n t e r e s t to i s o l a t e th i s metabolite i n s u f f i c i e n t l y large quantity to gather a d d i t i o n a l evidence for the proposed s t ruc ture . A v a i l a b i l i t y of a pure sample of the glucuronide w i l l help i n developing a d i r e c t assay method for the in tac t g lucuronide i n the u r i n e . Such methods can then be used for the study of s t e reose lec t ive g lucuron ida t ion . 4. While many antiarrhythmic drugs such as procainamide, l i d o c a i n e and mexi let ine have been measured i n the s a l i v a , the p o s s i b i l i t y of s a l i v a r y excret ion of toca in ide has never been i n v e s t i g a t e d . Therefore i t was of in te re s t to study the extent of excre t ion of toca in ide into s a l i v a and to determine i f a c o r r e l a t i o n existed between s a l i v a and plasma l e v e l s . In a d d i t i o n , the a p p l i c a t i o n of a s t e reose lec t ive assay for the determination of s a l i v a r y composition - 38 -of enantiomers would reveal i f there i s any s t e r e o s e l e c t i v i t y i n s a l i v a r y e x c r e t i o n . Such a p o s s i b i l i t y has never been inves t iga ted with c h i r a l drugs. 5 . Tocainide i s excreted i n the urine to the extent of 40-50% of the administered dose. Approximately 30% of the dose i s a lso excreted i n the urine as toca inide g lucuronide . Therefore toca inide e l imina t ion would depend on i n t e g r i t y of renal func t ion . It was of i n t e r e s t , there fore , to study the extent of accumulation of toca inide enantiomers i n pat ients with renal dysfunction and to determine i f the hemodialysis process removed the drug. - 39 -i EXPERIMENTAL A. Materials and Chemicals 1. Column Chromatography Amberlite XAD-2 r e s i n - Rohm & Haas C o . , P h i l a d e l p h i a , P a . , U . S . A . Acetone - Reagent grade, F i sher S c i e n t i f i c , Vancouver, B . C . , Canada. Methanol - HPLC grade, F i sher S c i e n t i f i c . E t h y l acetate - Burdick & Jackson Labrator ies I n c . , Muskegon, M i . , U . S . A . F r a c t i o n c o l l e c t o r - LKB Bromma f r a c t i o n c o l l e c t o r , Sweden. 2 . Thin-layer Chromatography 1,3-dihydroxynaphthalene (Naphthoresorcinol) - A l d r i c h Chemical C o . , Milwaukee, W i . , U . S . A . Phosphoric acid (85%) ' - Reagent grade, A l l i e d Chemical Company, Miss i s sauga , O n t . , Canada. Whatman K C i s F p lates - Whatman I n c . , C l i f t o n , N j . , U . S . A . S i l G-25 UV25I+ pre-coated plates - Brinkman Instruments (Canada) L t d . , Rexdale, O n t . , Canada. Methanol, A c e t o n i t r i l e , Chloroform - HPLC grade, F i scher S c i e n t i f i c . E t h y l acetate - Burdick & Jackson Laboratories Inc . Tetrahydrofuran, Ace t i c A c i d , Sodium Chlor ide - BDH (Canada) L t d . , Toronto, O n t . , Canada. TLC s treaking apparatus - Appl ied Science Laboratories L t d . , State C o l l e g e , P a . , U . S . A . 3 . High—performance L i q u i d Chromatography 5 u Ultrasphere ODS column (25 cm x 4.6 mm), Beckman Instruments I n c . , Berkeley , C a . , U . S . A . Water, A c e t o n i t r i l e , n-Hexane, Dichloromethane - HPLC grade, F i sher S c i e n t i f i c . - 40 -Gas-liquid Chromatography Heptaf luorobutyr ic anhydride - Pierce Chemical Company, Rockford, I I . , USA. Ether - reagent grade, A l d r i c h Chemical Company. Dimethyl su l foxide - F i sher S c i e n t i f i c . Sodium hydr ide , Calcium hydr ide , Methyl iodide - A l d r i c h Chemical Company. D-Glucuronic A c i d , p-Nitrophenol Glucuronide - Sigma Chemical Co. St . L o u i s , Mo. , U . S . A . C h i r a s i l - V a l ® glass c a p i l l a r y column (25 m x 0.25 mm) and C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.3 mm) -Appl ied Science. F u s e d - s i l i c a c a p i l l a r y column (uncoated), A l l t e c h Assoc ia te s , A r l i n g t o n Heights , I I . , U . S . A . Carbowax 20 M, A l l t e c h Assoc ia te s . S i l a r 10 C glass c a p i l l a r y column-coated i n the l abora tory . SP-2330 glass c a p i l l a r y column - Supelco, I n c . , B e l l e f o n t e , P a . , U . S . A . 0V-225 glass c a p i l l a r y column, - Appl ied Science Animal Surgery Ether solvent - USP grade, A l d r i c h Chemical Company. Heparin - Sigma Chemical"company. Polyethylene tubing (PE.50) - Beckton Dickinson & C o . , Parsippany New York, N . Y . , U . S . A . S i l a s t i c medical grade tubing - Dow Corning Corporation Medical Products , Midlands, M i . , U . S . A . Dermasept sk in cleanser - Germiphene Company L t d . , Brant ford , Ont Canada. - 41 -6. Miscellaneous 3-Glucuronidase (bovine l i v e r ) - (5000 units /mL, Sigma Chemical Company. Hydrochlor ic Acid - F i sher S c i e n t i f i c . Ammonium acetate - General Chemical D i v i s i o n , A l l i e d Chemical & Dye Corporat ion , New York, Ny, U . S . A . Sodium hydroxide - American S c i e n t i f i c and Chemical, Por t l and , O r . , U . S . A . Tocainide hydrochlor ide - As t ra Pharmaceutical Products , I n c . , Framingham, Ma . , U . S . A . l - A m i n o - 2 ' , 6 ' - a c e t o x y l i d i d e hydrochlor ide (W-49167) - As t ra Pharmaceutical Products . ct-Bromonaphthalene - ICN Pharmaceuticals I n c . , P la inv iew, New York, N y . , U . S . A . 3- (2 ,6-Xylyl )5-methylhydantoin - Astra Pharmaceutical Products . 2 ,6-Dimethylan i l ine - A l d r i c h Chemical Company. Carbobenzyloxy-D-alanine and N-tert « b u t o x y carbonyl-L-a lanine -Sigma Chemical Co. N ,N ' -Dicyc lohexy lca rbod i imide , (-f- )di-p-toluoyl-d-tartar ic acid monohydrate, 32% hydrogen bromide i n acet ic acid - A l d r i c h Chemical Company. Benzene, chloroform, ether - D i s t i l l e d i n Glas s , Caledon Laborator ies L t d . , Georgetown, O n t . , Canada. Monoethylg lyc inexyl id ide hydrochlor ide (MEGX) - Astra Pharmaceutical JProducts. B. Preparation of Reagents and Stock Solutions 1 . Tocainide Hydrochloride Solution ( 1 0 ug/mL) Tocanide hydrochlor ide (10 mg) was weighed accurate ly and d i s so lved i n d i s t i l l e d water i n a 10 ml volumetric f l a s k . An a l iquot of 1 mL of t h i s so lu t ion was d i l u t e d to volume with d i s t i l l e d water i n a 100 mL volumetr ic f l a s k . - 42 -2. W-49167 Hydrochloride Solution (10 ug/ml) l - A m i n o - 2 ' , 6 ' - a c e t o x y l i d i d e hydrochlor ide (10 mg) was weighed accurate ly and d i s so lved i n d i s t i l l e d water i n a 10 mL volumetr ic f l a s k . An a l iquot of 1 mL of th i s s o l u t i o n was d i l u t e d to volume with d i s t i l l e d water i n a 100 mL volumetr ic f l a s k . 3. a-Bromonaphthalene Solution (10 ug/mL) a-Bromonaphthalene (10 mg) was weighed accurate ly and d i s so lved i n benzene i n 10 ml volumetr ic f l a s k . An a l iquot of 1 mL of t h i s s o l u t i o n was d i l u t e d to volume with benzene i n a 100 mL volumetr ic f l a s k . 4. Monoethylglycinexylidide (MEGX) Hydrochloride Solution (10 ug/mL Monoethylg lyc inexyl id ide hydrochlor ide (10 mg) was weighed accurate ly and d i s so lved i n d i s t i l l e d water i n a 10 mL volumetr ic f l a s k . An a l iquot of 1 mL of th i s s o l u t i o n was d i l u t e d to volume with d i s t i l l e d water i n a 100 mL volumetr ic f l a s k . 5. Tocainide Base Solution (10 yg/mL) Tocainide hydrochlor ide (50 mg) was d i s so lved i n 5 mL d i s t i l l e d water and the s o l u t i o n was made a l k a l i n e by add i t ion of 1 mL of 1.0 N sodium hydroxide s o l u t i o n . This so lu t ion was extracted three times with 10 mL each of benzene. The benzene extracts were combined and evaporated under vacuum to dryness. The mater ia l was dr ied and stored i n a de s i cca to r . - 43 -Tocainide base (10 mg) was weighed accurate ly and d i s so lved i n 10 mL of benzene i n a 10 mL volumetric f l a s k . An a l iquot of 1 mL of t h i s s o l u t i o n was d i l u t e d with benzene to 100 mL to give a 10 ug/mL s o l u t i o n . 6 . Naphthoresorcinol Reagent Naphthoresorcinol (0.2 g) was d i s so lved i n a mixture of 165 mL of ethanol and 35 mL of phosphoric acid (85%). C. Preliminary Studies on C a p i l l a r y Column Gas Chromatography of Tocainide 1. Column Selection 1.1 Preparation of Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column A f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.2 mm) was coated with carbowax 20 M by the mercury plug dynamic method (Schomburg et a l . , 1974) as fo l lows : the c a p i l l a r y coating re se rvo i r was f i l l e d with a 2% s o l u t i o n of carbowax 20 M i n dichloromethane. The re se rvo i r also contained a small pool of mercury. One end of the c a p i l l a r y column was inser ted into the so lu t ion of the s ta t ionary phase through a septum-type sea l and the side-arm of the re servo i r was connected to a ni trogen source. The coating s o l u t i o n was forced through the column by ni t rogen pressure at a v e l o c i t y of 2 cm per sec . When 25% of the column length was f i l l e d with the s ta t ionary phase, the column end was lowered in to the pool of mercury to draw i n a 5 cm p lug . The s ta t ionary phase, along with the mercury plug was allowed to flow through the ent i re length of the column at a constant v e l o c i t y . In order to avoid a sudden change i n - 44 -coat ing v e l o c i t y as the s ta t ionary phase l e f t the column, the main column was connected to a buffer column of approximately 10 meters. When the l a s t drop of mercury l e f t the column, the flow of n i t rogen was increased to evaporate the so lvent . After drying under a stream of n i t rogen for three hours, the column was t ransferred to the oven of the gas chromatograph and one end of the column was connected to the i n j e c t i o n por t . The oven was temperature-programmed from 5 0 ° to 2 0 0 ° C at a rate of 1 ° per minute and the helium flow through the column was maintained at 1 mL per minute. The column was held at 2 0 0 ° C for 48 hours . Af ter c o n d i t i o n i n g , the performance of the column was evaluated by ana lys i s of a test-probe mixture containing nonane, decane, dodecane, d ibuty lketone , tetradecane, c i s -p ropy lcyc lohexano l , t rans-propylcyclohexanol and 2 , 6 - d i m e t h y l a n i l i n e . 1.2 Gas-liquid Chromatographic (GLC) Analysis of Tocainide on Carbowax  20 M Fused-silica Capillary Column In order to enhance the chromatographic propert ies of toca inide and to increase s e n s i t i v i t y of detect ion to low nanogram l e v e l s , toca in ide was converted to i t s hepta f luorobutyryl (HFB) d e r i v a t i v e as fo l lows : to 1.0 ug of toca in ide base d i s so lved i n 200 uL of n-hexane were added 30 uL of heptaf luorobutyr ic anyhydride. The t i g h t l y capped tubes were heated at 5 5 ° C for 30 minutes i n an aluminum block (Thermolyne D r i - B a t h , F i sher S c i e n t i f i c ) . The excess reagent and solvent were removed under a stream of ni trogen and the residue was recons t i tu ted i n 200 uL of n-hexane. A 1-2 yL a l iquot was used for the gas chromatographic (GC) a n a l y s i s . - 45 -Chromatographic condit ions were as fo l lows : i n j e c t i o n temperature, 2 4 0 ° C ; e l ec t ron capture detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 8 0 ° C ; c a r r i e r gas (helium) flow ra te , 1 mL/min; s p l i t vent f low, 100 mL/min; i n l e t pressure, 172.3 kPa, make-up gas (helium) f low, 50 mL/min. ; chart speed, 0.3 cm/min. 1.3 GC Analysis of Tocainide HFB Using C a p i l l a r y Columns Coated with  S i l a r 10 C, OV-225 and SP-2330 Chromatographic condit ions for ana lys i s of toca in ide heptaf luorobutyrate were the same as described before. 2. Determination of Optimum Condition f o r D e r i v a t i s a t i o n In order to determine the optimum time required for d e r i v a t i s a t i o n , s ix samples of toca inide base (1.0 ug each) were prepared i n 10 mL polyte tra f luoro-ethylene- (PTFE) l i n e d screw-capped cu l ture tubes and were heated with 30 uL of hepta f luorobutyr ic anhydride (HFBA) for 15, 30, 45, 60, 75 and 90 minutes at 5 5 ° C . The excess reagent and solvent (hexane) were removed as described before . The res idue was d i s so lved i n 200 uL of n-hexane containing 0.2 ug of a-bromonaphthalene and 1-2 uL of th i s s o l u t i o n was in j ec ted onto a carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column. A p lot of peak area ra t io s against time of d e r i v a t i s a t i o n revealed the optimum durat ion of d e r i v a t i s a t i o n . - 46 -3. Selection of Internal Standard a-Bromonaphthalene was used as an i n t e r n a l standard during the ear ly development of a g a s - l i q u i d chromatographic assay using packed-column and e l e c t r o n capture detect ion (Venkataramanan et a l . , 1978). Although th i s compound had good e l ec t ron capturing p r o p e r t i e s , i t was not found su i tab le as an i n t e r n a l standard i n the present technique because of i t s very short re tent ion time and lack of any s i m i l a r i t y to toca in ide . Monoethylg lyc inexyl id ide (MEGX), a metabolite of l i d o c a i n e which i s s t r u c t u r a l l y re la ted to t o c a i n i d e , reacts with HFBA to y i e l d i t s monoheptafluorpbutyrate d e r i v a t i v e . This compound has a longer re tent ion time than toca inide HFB and was chosen as the i n t e r n a l standard for the i n i t i a l s tud ies . 4. Check f o r S p l i t t e r D i f f e r e n t i a t i o n Between Tocainide and Internal  Standard, Monoethylglycine X y l i d i d e (MEGX) Compounds with widely varying molecular weights and v o l a t i l i t y are known to be d i f f e r e n t i a t e d by the glass in ser t used i n the i n j e c t i o n port for the s p l i t mode of i n j e c t i o n . To test for any d i f f e r e n t i a t i o n between toca in ide and the i n t e r n a l standard (MEGX), a mixture of t h e i r hepta f luorobutyry l der iva t ive s were analysed on a carbowax 20 M fused s i l i c a c a p i l l a r y column by varying the s p l i t r a t io s from 1:15 to 1:130. A c a r r i e r gas flow rate of 1 mL/min through the column was maintained throughout. - 47 -D. Pharmacokinetics of Tocainide i n the Rat Adult Wistar r a t s , with an average weight of about 250 ,g (216 to 378 jg) , . were used for the i n i t i a l study of the pharmacokinetics of intravenous toca inide i n th i s species (Rats were obtained from the Animal Care Unit of the U n i v e r s i t y of B r i t i s h Columbia). The animals were maintained i n m e t a l l i c cages i n a c o n t r o l l e d environment (12 hours day l ight ) for at leas t 3 days p r i o r to the experiments. Rat chow or lab chow was fed to the r a t s . They were fasted for a period of 8-10 hours p r i o r to , and during the experiments. Water was allowed ad l i b i t u m . 1. Plasma Level Study The plasma l e v e l study of toca inide i n the rat was c a r r i e d out using an implanted jugular ve in cannula for blood sampling. The cannulat ion procedure used was a minor modi f i ca t ion of the technique developed by Weeks and Davis (1964). 1.1 Preparation of Jugular Vein Cannula The jugular ve in cannula consisted of polyethylene tubing (PE-50) jo ined to a s i l a s t i c tubing by means of a short length of hypodermic needle . The hypodermic needle, about 4 mm long , was inser ted i n the polyethylene tubing to a distance of about 2 mm. The s i l a s t i c tubing was then threaded over the metal connector and pos i t ioned over the sec t ion of polyethylene covered metal tubing . The s i l a s t i c was secured i n place by 4-0 s i l k thread. The cannula end of the polyethylene tubing was then shaped into a " U " by bending around a pasteur p ipet te and dipping momentarily in to - 48 -b o i l i n g water. The cannula was cleaned with d i s t i l l e d water, soaked i n 95% a lcohol for 3-5 hrs and stored i n s t e r i l e normal s a l ine s o l u t i o n p r i o r to use. 1.2. S u r g i c a l Implantation of the Cannula The rat was anesthetised with ether i n a des iccator j a r . Af ter removing the rat from the de s i cca tor , the anesthesia was maintained throughout the implantat ion procedure by p lac ing ether-impregnated cotton close to the nose of the r a t . The ha i r on the'back of the animal was c l ipped and a point was marked at the center of the back of the neck to ind ica te the e x t e r i o r i z a t i o n point for the cannula. The rat was then placed on i t s back and the ha i r on the area over the r i gh t external jugular ve in was c l i p p e d . This area was recognized by observing the rap id pu l sa t ion of the jugular v e i n . The shaved area was cleansed by wiping with a s t e r i l e gauze saturated with 70% a l c o h o l . A l o n g i t u d i n a l i n c i s i o n , about 2 cm long , was made on the sk in over the jugular ve in and the ve in was exposed by debriding the surrounding f a s c i a . The v e i n was l i g a t e d at the upper end using a s i l k suture . A probe was inser ted subcutaneously ju s t behind the ear from the i n c i s i o n on the neck to the back sk in i n c i s i o n . The cannula, f i l l e d with hepar ini sed sa l ine (20 uni t s /mL) , was then inserted into the probe and the probe was pul led towards the jugular v e i n . I f the cannula was located i n the correct plane, the ' U ' port ion l a i d f l a t on the neck muscle. A probe was then placed under the v e i n , ra i sed s l i g h t l y and a small puncture was made into the ve in w a l l . The t i p of the s i l a s t i c - 49 -tubing was cut in to a 4 5 ° bevel so as to f a c i l i t a t e easy i n s e r t i o n of the cannula into the v e i n . Inser t ion of the cannula in to the puncture hole was further f a c i l i t a t e d by gently l i f t i n g the l i p of the hole with a pa i r of f ine forceps and i n s e r t i n g the cannula into the blood ves se l wi th a slow, gentle r o t a t i o n of the t i p . The s i l a s t i c tubing was then advanced slowly up to the j u n c t i o n of the s i l a s t i c - p o l y e t h y l e n e tubing and then secured i n the ve in with a 4-0 s i l k suture. The cannula was anchored i n p o s i t i o n by ty ing the suture (used to secure the tubing i n the vein) to the adjacent muscle. The subcutaneous connective t i s sue was reunited with s u r g i c a l thread and the sk in i n c i s i o n was closed using a 0-0 s i l k thread. The protruding end of the cannula was then brought subcutaneously to the back of the neck and secured i n p o s i t i o n by c l o s i n g the sk in i n c i s i o n with 0-0 s i l k . The cannula was checked to ensure that i t was f u n c t i o n a l . About 4 cm of cannula was allowed to protrude from the nape of the neck. The cannula was sealed by i n s e r t i n g a p in into the open end. P e r i o d i c use of heparinised s a l ine (20 units/mL) helped to keep the cannula f u n c t i o n a l . 1.3 Drug Administration and S e r i a l Blood C o l l e c t i o n About 0.2 mL of heparinized sa l ine (20 units/mL) was taken i n a 1 mL syringe with a 23 G needle and 0.2 mL of blood was withdrawn from the animal v i a the jugular ve in cannula. Subsequently, 0.5 mL of aqueous so lu t ions of toca in ide hydroc lor ide ( i n normal sa l ine ) corresponding to 15, 20 or 25 mg/kg were in jec ted through the jugular ve in cannula. The cannula was then flushed with the contents of the f i r s t syringe (0.2 mL - 50 -blood + 0.2 mL heparinised s a l i n e ) , thus the rat was ensured of r e c e i v i n g the ent i re dose. At appropriate time i n t e r v a l s (3, 6, 10, 15, 20, 30, 45, 90, 120, 150, 180, 240, 300, 360, 420, 480 min.) a syringe with 0.2 mL of hepar inised sa l ine was introduced into the cannula and about 0.15 mL of the f l u i d was withdrawn. A fresh needle and syringe was introduced and about 0.25 mL of blood was withdrawn. The so lu t ion i n the f i r s t syringe (contents from cannula, some blood and heparinised sa l ine) was r e in j ec ted into the animal . The blood samples were t ransferred to heparinised Caraway® tubes and one end was sealed using a C r i t o c a p ® . The tubes were centri fuged for 10 minutes at 2500 r . p . m . and the separated plasma samples were kept frozen u n t i l analysed. 2. Urinary Excretion Studies: Tocainide-dosed animals were housed i n s t a in l e s s s tee l metabolism cages with f a c i l i t i e s for urine c o l l e c t i o n free of f eca l contamination. Af ter 24 hours , the sides of the cage were washed with d i s t i l l e d water into an amber colored urine c o l l e c t i o n b o t t l e . Sui table d i l u t i o n s of the urine sample thus c o l l e c t e d were made and kept frozen u n t i l analysed. E . Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n f o r Rat Plasma  and Urine Assay To f i v e 100 uL a l iquot s of ra t plasma or ur ine obtained from untreated male Wistar r a t s , were added 50, 100, 200, 500, and 1000 ng of - 51 -toca in ide hydrochlor ide from an aqueous s o l u t i o n (1.0 pg/mL). An a l iquot of the i n t e r n a l standard so lu t ion equivalent to 1.0 pg of monoethylg lyc inexyl id ide (10.0 pg/mL) was added to each tube, followed by 200 pL of 1.0 N sodium hydroxide. T r i p l i c a t e samples of each concentrat ion were prepared. Five m i l l i l i t e r s of dichloromethane were added to each tube and the tubes were shaken for 10 minutes on a rotary shaker. Af ter cent r i fuga t ion at 2500 rpm for 10 minutes, 4 mL of the organic phase were transferred to 15 mL po lyte t ra f luoroethylene l i n e d screw-capped centr i fuge tubes and the contents of the tubes evaporated i n a water both at 4 0 ° C under a gentle stream of n i t rogen . To the residue were added 100 uL of n-hexane and 30 pL of hepta f luorobutyr ic anhydride. The tubes were t i g h t l y capped and heated at 5 5 ° C for 30 minutes i n an aluminum block . The excess reagent was removed by slow evaporation with a stream of n i t rogen and the residue was d i s so lved i n 200 pL of n-hexane; 1-2 pL of th i s so lu t ion was used for gas chromatographic a n a l y s i s . F. Resolution of Tocainide Enantiomers on C h i r a s i l - V a l 8 Glass C a p i l l a r y  Column To 1.0 pg of ( ± ) toca in ide hydrochlor ide contained i n 10 mL screw-capped cu l ture tubes (0.1 mL of a stock so lu t ion conta ining 10 pg/mL) were added 200pL of 1.0 N sodium hydroxide s o l u t i o n and 5 mL of dichloromethane. The tubes were t i g h t l y capped and shaken for 10 min on a ro tary shaker. The dichloromethane was separated to a c l e a n , dry cu l ture tube and the contents were evaporated to dryness under a stream - 52 -of n i t rogen . To the dry residue was added 200 uL of n-hexane and the contents of the tube were mixed by vortexing for a few seconds. Hepta f luorobutyr ic anhydride (30 uL) was then added to the tube and i t was t i g h t l y capped with a po lyte t ra f luoroethylene (PTFE)- l ined screw-cap and heated at 5 5 ° C i n an aluminum block for 30 minutes. The tube was cooled to room temperature and the excess reagent was removed by evaporation with a stream of n i t rogen u n t i l the odour of HFBA was not apparent. To the residue was added 200 uL of n-hexane and 1 \iL of th i s s o l u t i o n was in jec ted onto the C h i r a s i l - V a l ® glass c a p i l l a r y column. A model 5830A gas chromatograph (Hewlett Packard) equipped with a s p l i t mode i n j e c t i o n port and a 6 3 N i e lec t ron capture detector was used for the chromatographic a n a l y s i s . G. Synthesis of Tocainide Enantiomers 1. Synthesis of R(-) Tocainide Hydrochloride To a s o l u t i o n of 13.38 g (0.06 mole) of carbobenzyloxy-D-alanine and 7.3 g (0.06 mole) of 2 ,6 -d imethy lan i l ine i n 150 ml of d i c h l o r o -methane were added 13.6 g (0.066 mole) of N ,N ' -d icyc lohexylcarbodi imide i n 60 mL of dichloromethane. Af ter the so lu t ion was l e f t standing at room temperature for one hour, the p r e c i p i t a t e d N,N ' -d icyc lohexylurea was f i l t e r e d off and the solvent was evaporated from the f i l t r a t e under reduced pressure leaving 12.3 g of white s o l i d , m.p. 1 6 7 - 1 6 8 ° C . To remove the carbobenzyloxy group, 70 mL of a s o l u t i o n of 30-32% hydrogen bromide i n ace t i c ac id were added to 12.3 g of the above reac t ion - 53 -product and the mixture was s t i r r e d u n t i l d i s s o l v e d . To th i s s o l u t i o n , 200 ml of dry d ie thy le ther were added and the p rec ip i t a t ed tocainide hydrobromide was f i l t e r e d off and d r i e d , y i e l d i n g 8.2 g of white s o l i d , m.p. 2 6 7 ° C . This mater ia l was converted to a hydrochloride s a l t with e thanol i c hydrogen ch lor ide s o l u t i o n and r e c r y s t a l l i z e d from e t h a n o l - d i e t h y l ether to y i e l d R(-) tocainide hydrochlor ide , m.p. 2 6 5 - 2 6 6 ° C and ( a ) p 5 - 4 2 . 1 6 ° (c , 2.63 i n methanol). A sample of this mater i a l was converted to i t s d e r i v a t i v e with hepta f luorobutyr ic anhydride as described before. The pur i ty was determined by gas chromatographic analys i s on a C h i r a s i l - V a l ® column. The product synthesized had an enantiomer r a t i o of 95:5 for the R and S enantiomers, r e s p e c t i v e l y . 2. Synthesis of S(+) Tocainide Hydrochloride To a so lu t ion of 18.9 g (0.1 mole) of N-ter t *butoxycarbonyl-L-alanine and 12.1 g (0.1 mole) of 2 ,6 -d imethy lan i l ine i n 200 ml of dichloromethane were added 20.6 g (0.1 mole) of N , N ' - d i c y c l o h e x y l -carbodimide. Af ter the mixture was s t i r r e d at room temperature for 1 hour, the p rec ip i t a t ed N , N ' - d i c y c l o h e x y l urea was f i l t e r e d off and the solvent was evaporated from the f i l t r a t e under reduced pressure, l eav ing 16.1 gm of a creamy white s o l i d , m.p. 1 3 1 ° C . To remove the N-ter t •butoxycarbonyl group, 50 mL of 30-32% hydrogen bromide i n ace t ic acid were added to 10 g of the above reac t ion product and the mixture was s t i r r e d u n t i l d i s s o l v e d . To this so lu t ion 200 mL of dry d ie thy le ther were added and the prec ip i t a ted tocainide hydrobromide was f i l t e r e d and d r i e d , y i e l d i n g 7.8 g of white s o l i d , - 54 -m.p. 2 7 4 ° C . This mater ia l was converted to a hydrochlor ide s a l t and r e c r y s t a l l i z e d from e thano l -d ie thy l ether to y i e l d S(+) toca in ide hydroch lor ide , m.p. 2 6 6 ° C . A sample of th i s mater ia l was analysed as i t s hepta f luorobutyry l d e r i v a t i v e on the C h i r a s i l - V a l ® column and was found to cons i s t of 81:19 r a t i o of S:R isomers. To increase the o p t i c a l p u r i t y of th i s product, toca inide base, obtained from 1 g of the mater i a l was added to a warm so lu t ion of 1.4 g of d i - p - t o l u o y l - 1 -t a r t a r i c acid i n 10 mL of 95% ethanol . The dias tereoisomeric sa l t was c r y s t a l l i z e d 5 times at room temperature to y i e l d f ine needles . This mater ia l was converted to i t s hydrochlor ide sa l t and r e c r y s t a l l i z e d from e t h a n o l - d i e t h y l ether to y i e l d S(+) toca inide hydroch lor ide , m.p. 2 6 6 ° C , 2 5 [ c t ] D + 4 2 . 3 5 ° (c 2.63 i n methanol) . An evaluat ion of the enantiomer r a t i o of the hepta f luorobutyryl d e r i v a t i v e on the C h i r a s i l - V a l ® column ind ica ted a r a t i o of 91:9 of the S:R isomers. 3. Determination of o p t i c a l p u r i t y and Id e n t i t y of S(+) and R(-)  tocainide by gas chromatograpby/mass spectrometry (GCMS) In order to determine the composition of the product synthes ized, a GCMS technique using C h i r a s i l - V a l ® glass c a p i l l a r y was employed. The GCMS ana lys i s of the hepta f luorobutyryl de r iva t ive s of (R ,S ) -toca inide was done under the fo l lowing c o n d i t i o n s : i n j e c t i o n temperature, 2 4 0 ° C ; oven temperature, 5 0 ° C to 1 5 0 ° C at 3 0 ° C / m i n , 1 5 0 ° C to 2 0 0 ° C at 5 ° C / m i n ; s p l i t l e s s mode of i n j e c t i o n , source temperature, 2 0 0 ° C ; analyser temperature, 2 4 0 ° C ; t ransfer l i n e temperature, 2 4 0 ° C , i o n i s a t i o n p o t e n t i a l , 70 ev. - 55 -4. Measurement of O p t i c a l Rotation of Tocainide Enantiomers O p t i c a l r o t a t i o n studies were conducted on a Perkin-Elmer Model 142 polarimeter (Perkin-Elmer, Norwa^lk, C t . , USA) i n a 0.1 dm tube at 2 5 ° C . Tocainide hydrochlor ide (131.5 mg) was accurate ly weighed and d i s so lved i n 5 mL of methanol (HPLC grade) . The c e l l of the polar imeter was f i l l e d with th i s s o l u t i o n and the o p t i c a l r o t a t i o n was measured. The s p e c i f i c r o t a t i o n of S(+)tocainide hydrochlorde (c , 2.63% i n MeOH) 25 was ( a ) D = (+) 4 2 . 3 5 ° and the s p e c i f i c r o t a t i o n of R(-) toca in ide hydrochlor ide ( c , 2.63 i n MeOH) was ( a ) " = (-) 4 2 . 1 6 ° . H. 1. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Assay  of Tocainide Enantiomers i n Human Plasma and Urine To 0.5 mL of plasma were added 100, 250, 500, 750, 1000, and 1500 ng of racemic toca inide hydrochlor ide from a stock so lu t ion and 1000 ng of the i n t e r n a l standard. In a s i m i l a r fashion 100, 200, 400, 1000, and 2000 ng of racemic toca inide were added to 0.1 mL of urine along with 1000 ng of i n t e r n a l standard. T r i p l i c a t e samples of plasma and urine thus prepared were extracted and d e r i v a t i s e d as described before . 2. Inter- and Intra-Assay V a r i a t i o n s To 0.1 mL of u r i n e , contained i n three cu l ture tubes, were added 100 uL of ( ± ) t o c a i n i d e hydrochlor ide so lu t ion (stock s o l u t i o n , 10 pg/mL), 100 uL of monoethylg lyc inexyl id ide hydrochlor ide s o l u t i o n (stock s o l u t i o n , 10 ug/mL), 200 uL of 1.0 N sodium hydroxide so lu t ion and - 56 -5 mL of dichloromethane. The tubes were t i g h t l y capped and tumbled on a tube tumbler for 10 minutes. The dichloromethane was separated into three clean dry cul ture tubes and the solvent was removed by evaporat ion with a stream of n i t rogen . To the residue were added 200 pL of n-hexane and 30 pL of hepta f luorobutyr ic anhydride. The tubes were t i g h t l y capped and heated i n an aluminum block at 5 5 ° C for 30 minutes. The excess reagent was removed by slow evaporation at room temperature with a stream of ni trogen and the residue was reconst i tuted i n 200 pL of n-hexane. An a l iquot of 1 pL of this so lu t ion was in jec ted onto the C h i r a s i l - V a l ® glass c a p i l l a r y column and the peak area r a t i o s of drug to i n t e r n a l standard were determined. Three i n j e c t i o n s were made from each tube and the peak area r a t io s measured were used to compute v a r i a t i o n s between i n j e c t i o n s of the same sample as wel l as v a r i a t i o n s between these samples. I. Preliminary Study of S t e r e o s e l e c t i v i t y of Tocainide D i s p o s i t i o n i n  the Human Two healthy male volunteers (38 years , 68 kg and 39 years , 98 kg) , fasted overnight for 12 h , were administered 100 mL of an o r a l s o l u t i o n of racemic tocainide hydrochlor ide at a dose of 3 mg/kg body weight under the supervis ion of a phys i c i an . Food was allowed a f ter three hours and water was allowed _ad l i b i t u m . An indwel l ing b u t t e r f l y cannula was inserted i n the c u b i t a l ve in of the arm and was used to c o l l e c t blood samples for the f i r s t 3 hours. Subsequent blood samples were c o l l e c t e d by venipuncture. Blood samples (8 mL) were co l l ec ted at - 57 -15 minute i n t e r v a l s for the f i r s t 2 hours and at 3, 5, 7, 24, 48, and 72 hours therea f ter . Blood samples were centri fuged and the separated plasma was stored at - 2 0 ° C u n t i l assayed. Urine samples were c o l l e c t e d i n polyethylene bags at 0, 1, 2, 3, 5, and 7 hours and thereafter at the convenience of the subject and at blood withdrawal times up to 96 hours. Samples were kept frozen u n t i l analysed. Samples of plasma and ur ine were also obtained before drug inges t ion to serve as blanks and for the determination of c a l i b r a t i o n curve data and p r e c i s i o n . J . 1. Comparison of the A n a l y t i c a l Results Obtained f o r Racemic Tocainide Using a Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column  with those of Tocainide Enantiomers Using the Chirasil-Val®  Glass C a p i l l a r y Column In order to compare the values obtained by analys i s of the t o t a l racemate, the same samples from one volunteer were also analysed by a non-s tereospec i f i c c a p i l l a r y column gas chromatographic method employing f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.2 mm) coated with carbowax 20 M. 2. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Human  Plasma Assay by Carbowax 20 M F u s e d - s i l i c a C a p i l l a r y Column Human plasma (0.5 mL), to which 50 ng to 1000 ng of ( ± ) toca in ide hydrochlor ide along with 1000 ng of i n t e r n a l standard (MEGX) had been added, were extrac ted , d e r i v a t i s e d and analysed as described before . The r e l a t i v e standard dev ia t ion of the peak area r a t io s for t r i p l i c a t e samples of each concentrat ion were determined. - 58 -K. Determination of C a l i b r a t i o n Curve Data and Assay P r e c i s i o n 1. Human Plasma and Urine Assay Employing a Chirasil-Val® F u s e d - s i l i c a  C a p i l l a r y Column and W-49167 as In t e r n a l Standard To 0.5 mL of blank plasma or 0.1 mL of blank ur ine were added 25 ng to 2000 ng of ( ±) tocainide hydrochlor ide and 1000 ng of the i n t e r n a l standard, 1-aminoacetoxylidide. These samples were extracted , d e r i v a t i s e d and analysed as described before. 2. Confirmation of the Structure of the Ac y l D e r i v a t i v e of 1-aminoacetoxylidide (W-^49167) by C a p i l l a r y GCMS To 1.0 pg of W-49167 hydrochlor ide i n aqueous s o l u t i o n were added 200 pL of 1.0 N sodium hydroxide so lu t ion and 5 mL of dichloromethane. The mixture was shaken for 10 minutes on a tube tumbler. The dichloromethane was separated, evaporated and the residue d e r i v a t i s e d with hepta f luorobutyr ic anhydride as described before. This sample was analysed using GCMS and both e lec t ron impact (E l ) and chemical i o n i z a t i o n (CI) spectra were obtained. L . E f f e c t of Sodium Hydroxide Treatment on Urine Containing Tocainide  Carbamoyl-O- fr-D-glucuronide I t has been reported that ester glucuronides are unstable i n a l k a l i n e so lut ions and that they are r e a d i l y hydrolysed thus generating the parent compound (Hasegawa et a l . , 1982). Since the assay procedure involved add i t ion of sodium hydroxide so lu t ion (200 pL of 1.0 N so lut ion) to l i b e r a t e the free base from i t s s a l t s present i n the u r i n e , - 59 -i t was important to e s t a b l i s h that no tocainide was generated from i t s glucuronide during the assay procedure. Pure diphenhydramine (base) was used as an i n t e r n a l standard i n this experiment for the GLC-determina-t ion of tocainide i n ur ine using a short SE-30 (methyl s i l i c o n e ) f u s e d - s i l i c a c a p i l l a r y column (15 m x 0.25 mm) with flame i o n i s a t i o n d e t e c t i o n . Toca in ide , as the free base, e luted from this column with a r e t e n t i o n time of 4 minutes and the i n t e r n a l standard eluted at 2.6 minutes (column temperature 1 6 0 ° ) . Procedure An a l i q u o t of 0.1 mL of urine conta ining high proport ions of tocainide glucuronide (15 hour urine sample from one of the v o l u n t e e r s ) , was dispensed into 5 test tubes and 0.5 mL of a buffer so lu t ion (pH, 7.0 - 13.0) was added to each tube. A buffer so lu t ion (0.2 M) conta ining bor ic a c i d , potassium c h l o r i d e , and sodium hydroxide was used to adjust the pH of the u r i n e . The contents were extracted with 5 mL of methylene d i c h l o r i d e and evaporated to dryness by a gentle stream of n i t rogen . To the residue were added 50 uL of diphenhydramine so lu t ion (1 mg/mL i n CH3OH) and the samples were mixed on a vortex mixer. An a l iquot of 1-2 uL of th i s so lu t ion was used for gas chromatographic analys i s under the fo l lowing condi t ions : i n j e c t i o n temperature = 2 4 0 ° C ; column temperature = 1 6 0 ° C ; c a r r i e r gas (He) flow rate = 1 mL/min; s p l i t vent flow = 30 mL/min; make-up gas (He) = 50 mL/min. - 60 -M. Pharmacokinetics of Tocainide Enantiomers i n Healthy Subjects 1. Intravenous Administration of Racemic Tocainide Hydrochloride Five healthy male volunteers (age, 38-42 years ; weight, 65-98 kg) fasted for 12 hours overnight , were administered 200 mg of R ,S- toca in ide hydrochlor ide intravenously as an in fu s ion at a rate of 10 mg/min for 20 minutes under medical superv i s ion . Electrocardiograms were recorded p r i o r to , and 1 and 2 hours a f ter the drug i n f u s i o n . No food was allowed for 3 hours p o s t - i n f u s i o n . Blood samples (8 mL) were c o l l e c t e d i n t o hepar in ized V a c u t a i n e r s ® (Becton Dickinson & Co.) through a b u t t e r f l y cannula inser ted i n the b r a c h i a l ve in at 0 .3 , 0 .5 , 0 .75, 1.0, 1.5, 2 .0 , 3, 5, 7, 10, 24, 30, 48, and 72 hours. Unstimulated s a l i v a samples (2 mL) were c o l l e c t e d into 5 mL glass v i a l s at the same time as blood samples and the pH was immediately measured. Urine samples were also c o l l e c t e d at 1.0, 2 .0 , 3 .0 , 5 .0 , 7 .0 , 10.0, 24 .0 , 48 .0 , 72.0 and 96.0 hours and at the convenience of the subject at other t imes. The pH of the urine as w e l l as i t s volume was measured immediately a f ter c o l l e c t i o n . Urine was stored at - 2 0 ° C u n t i l required for a n a l y s i s . The thawed ur ine was centri fuged at 2000 r . p . m . for 10 minutes and a 100 uL a l iquot was used for ex t rac t ion of toca inide enantiomers as described before . 2. Oral Administration of Racemic Tocainide Hydrochloride Tocainide hydrochlor ide tab le t s (200 mg) were given to seven healthy male volunteers who were fasted overnight . Blood samples (8 mL) were c o l l e c t e d into hepar inised V a c u t a i n e r s ® through a b u t t e r f l y cannula - 61 -inser ted i n the b r a c h i a l v e i n at 0.25, 0 .5 , 0.75, 1.0, 1.25, 1.5, 1.75, 2 .0 , 3 .0 , 5 .0 , 7 .0, 10.0, 24.0, 36.0, 48 and 72 hours. Urine was c o l l e c t e d at 1.0, 2 .0 , 3.0, 5 .0 , 7 .0 , 10.0, 24.0, 48.0, 72.0 and 96 hours and at the convenience of the subject at other times. The pH of the ur ine as wel l as i t s volume was measured immediately a f ter c o l l e c t i o n . Urine was stored at - 2 0 ° C u n t i l required for a n a l y s i s . The thawed urine was centri fuged at 2000 r . p . m . for 10 minutes and a 100 uL a l iquot was used for ex t rac t ion of toca inide enantiomers as described before . N. 1. Assay of Tocainide Hydrochloride Tablets Tocainide hydrochlor ide tab le t s were weighed (10 tab le t s ) and an average weight was determined. A powdered sample, equivalent to 50 mg of toca in ide hydroch lor ide , was d i s so lved i n d i s t i l l e d water contained i n a 50 mL volumetric f l a s k . One m i l l i l i t e r of t h i s so lu t ion was d i l u t e d to 100 mL with d i s t i l l e d water to give a 10 ug/mL s o l u t i o n , from which 0.1 mL (1 yg) was used for the assay. T r i p l i c a t e samples were analysed under i d e n t i c a l cond i t ions . 2. Assay of Tocainide Hydrochloride I n j e c t i o n From a 5 mL ampoule (50 mg/ml), 0.2 mL of the i n j e c t i o n was p ipet ted out and d i l u t e d to 10 mL with d i s t i l l e d water. From t h i s stock s o l u t i o n (1 mg/mL) a d i l u t e so lu t ion containing 10 ug/mL was prepared and 0.1 mL of t h i s so lu t ion was used for the assay. T r i p l i c a t e samples were analysed and the area r a t i o s compared to that of 1 yg of standard toca inide hydrochlor ide processed i n the same manner. - 62 -3. Determination of Adsorption of Tocainide by the P l a s t i c Tubing of  the Infusion Set In order to ru le out adsorption of the drug by the p l a s t i c tubing used for the in fus ion set , toca inide hydrochlor ide i n j e c t i o n was assayed before and after passing through the in fus ion set . The contact time of the so lu t ion with the p l a s t i c tubing was 20 minutes. These samples were assayed as the racemate using a carbowax 20M f u s e d - s i l i c a c a p i l l a r y column (20 M x 0.2 mm). Three samples for analys i s were prepared as described under assay of i n j e c t i o n . 0. 1. Chromatographic Analysis of Uremic Plasma Extract on a  Chirasil-Val® F u s e d - s i l i c a C a p i l l a r y Column Plasma obtained from a pat ient who was anephric (serum c r e a t i n i n e , 13.4 mg%) was extracted , d e r i v a t i s e d and analysed on a C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column under the condi t ions described before . 2. Chromatographic Analysis of Uremic Plasma Extract on a Carbowax 2 0 M  F u s e d - s i l i c a C a p i l l a r y Column Plasma samples containing varying amounts of c r e a t i n i n e (4.9 to 16.8 mg%) (obtained from Vancouver General Hospi ta l ) were extracted , d e r i v a t i s e d and analysed on a carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.2 mm) as described before . - 63 -3. Chromatographic Analysis of Uremic Plasma Extracts on a Dual  C a p i l l a r y Column (Carbowax 20 M and Chirasil-Val®) In order to separate i n t e r f e r i n g peaks due to endogenous m a t e r i a l , two columns were connected together by a column-coupler (Chromatographic S p e c i a l i t i e s , B r o c k v i l l e , O n t a r i o ) . Plasma samples containing 13.4 mg% crea t in ine were analysed as descr ibed before, employing c a p i l l a r y columns c o n s i s t i n g of a carbowax 20 M f u s e d - s i l i c a column (50 m x 0.2 mm) on the i n j e c t o r side and a C h i r a s i l - V a l ® f u s e d - s i l i c a column (0.50 m x 0.3 mm) on the detector s ide . This combination of columns was used for the analys i s of plasma c o l l e c t e d from a pat ient who was anephric and who had been given 200 mg of ( ± ) tocainide hydrochlor ide by intravenous i n f u s i o n . 4. Preliminary Study of Tocainide Enantiomer D i s p o s i t i o n i n an Anephric P a t i e n t 4.1 Intravenous Administration of Racemic Tocainide Hydrochloride Racemic tocainide hydrochlor ide was administered by constant rate intravenous in fus ion (10 mg/min) for 20 minutes to a pat ient (male, age, 23 years; weight, 75 kg) with a serum crea t in ine l e v e l of 13.4 mg%. This pat ient had been undergoing hemodialysis three times a week for 2 years . Blood (1 mL) was withdrawn at 0 .33, 0 .5 , 0.75, 1.0, 2 .0 , 3 .0 , 5 .0 , 7 .0 , 10.0, 12.0 and 24 hours pos t - in fus ion into a glass syringe through a b u t t e r f l y cannula inserted i n the brach ia l v e i n . Blood was immediately transferred to cu l ture tubes and centri fuged at 1000 g for 10 minutes and the plasma was separated and kept frozen - 64 -at - 2 0 ° C u n t i l analysed. Sa l iva (1 mL) was also c o l l e c t e d into 5 mL glass v i a l s at the same time as plasma c o l l e c t i o n , the pH was measured, and the samples were kept frozen at - 2 0 ° C u n t i l required for the assay. 4.2 Hemodialysis Twenty-four hours af ter the intravenous in fu s ion of racemic toca inide hydroch lor ide , hemodialysis was started for a period of 5 hours using a hollow f i b e r d i a l y s e r (CF 1211) with a cuprophan membrane (surface area 0.8 m ) . The blood flow rate was 200 mL/min and the d i a ly sa te flow rate was 500-550 mL/min. Both a r t e r i a l and venous blood samples (1 mL) were c o l l e c t e d at one hour i n t e r v a l s during the d i a l y s i s period for the determination of toca in ide l e v e l s . P. Determination of C a l i b r a t i o n Curve Data and P r e c i s i o n of Assay  of S a l i v a The frozen s a l i v a samples were thawed and centrifuged at 2000 r . p . m . for 10 minutes to remove p a r t i c u l a t e matter. To 0.5 mL of the c l ea r supernatant s a l i v a contained i n a 10 mL cu l ture tube were added 100, 200, 400, 1000, 1500 and 3000 ng of racemic t o c a i n i d e , along with 1000 ng of the i n t e r n a l standard (W-49167). Subsequently, 200 yL of 1.0 N sodium hydroxide so lu t ion and 5 mL of dichloromethane were added and the toca in ide enantiomers were extracted , d e r i v a t i s e d and analysed as described previous ly under ana lys i s of plasma and u r i n e . - 65 -Q. Analysis of Tocainide Metabolites i n the Urine 1. I s o l a t i o n of Glucuronides from Urine by Adsorption on XAD-2 Resin  Column F i f t y grams of amberlite XAD-2 r e s i n were packed i n a 3 x 60 cm glass column. The column was washed with 200 mL of acetone, followed by 500 ml of d i s t i l l e d water. Subsequently, 200 mL of urine (obtained from a healthy volunteer who had been dosed with 200 mg of ( ± ) toca in ide hydrochlor ide) were percolated through the r e s i n column at a rate of 3 mL/min. The column was then washed with 500 mL of d i s t i l l e d water to remove unbound substances (urea , amino ac id s , inorganic sa l t s ) and then eluted with 600 mL of methanol. The eluate was c o l l e c t e d i n 5 mL f r ac t ions using a f r a c t i o n c o l l e c t o r . Every 5th f r a c t i o n was then tested for the presence of glucuronides by spott ing the sample on a s i l i c a ge l t h i n - l a y e r chromatography (TLC) p l a t e , spraying with 0.1% naphthoresorcinol i n ethanol/phosphoric a c i d , followed by heating the plates at 1 3 0 ° C for 10 minutes. The appearance of a blue colour was taken as an i n d i c a t i o n of the presence of g lucuronides . (p-Nitrophenol g lucuronide , treated i n an i d e n t i c a l manner gave a blue c o l o u r ) . The methanolic f r ac t ions containing the glucuronides were pooled and the solvent was removed by vacuum evaporat ion. The aqueous residue was adjusted to pH 3 with 1.0 N-hydrochlor ic acid and washed with three , 60 mL port ions of hexane. The aqueous phase was then f reeze-dr ied and the residue was recons t i tu ted i n methanol. This p a r t i a l l y p u r i f i e d methanolic extract was used for further analys i s by t h i n - l a y e r chromatography. - 66 -2. Thin-layer Chromatographic Separation of the Glucuronides Methanolic extract of the crude urine sample (5 uL) prev ious ly described was appl ied to a TLC plate (Whatman K C i s , 5 x 20 cm) along with a standard g lucuronide , p-Nitrophenol glucuronide (5 pL of a 1 mg/mL s o l u t i o n ) . Six such plates were developed i n the fo l lowing solvent systems: 1. 25% methanol/75% water 2. 50% methanol/50% water 3 . 60% methanol/40% water 4 . 75% methanol/25% water 5. 90% tetrahydrofuran/10% water 6. 75% acetoni tr i le /25% water For solvents conta ining more than 40% water, an amount of sodium c h l o r i d e s u f f i c i e n t to give a a 0.5 M concentrat ion was added. The plates were developed i n 6 x 22 cm c i r c u l a r glass tanks. The solvent was allowed to run to 15 cm on the p l a t e . The plates were then sprayed with naphthoresorcinol reagent and heated at 1 3 0 ° for 10 mins. Out of the s ix solvent systems tested, the combination of 60% methanol 40% water was found to be the most optimal solvent system for separating the crude methanolic ur ine ex t rac t . 3. Preparative Thin-layer Chromatographic I s o l a t i o n of  Tocainide Glucuronides An a l i q u o t of 150 pL of the crude methanolic extract.was appl ied - 67 -i n a narrow band to s ix 20 x 20 cm KCjg p lates and was developed with methanol/water (60:40) solvent system. To observe the band separation and to avoid heating the whole p l a t e , a 1 cm width of glass was cut from both ends of the plate with a diamond c u t t e r . The s t r i p s of glass were l a b e l l e d , sprayed with naphthoresorcinol reagent and heated i n an oven at 1 3 0 ° for 10 mins. The glass s t r ip s were then al igned with the unsprayed por t ion of the plate to locate the separated bands. Bands corresponding to most intense blue colour were scraped off the remainder of the p l a te s , and these were extracted from the support mater ia l with methanol and centr i fuged . The c lear methanolic extract was concentrated and re-appl ied to a second C^g plate (20 x 20 cm) as a narrow band. The samples were eluted with methanol:water (50:50) solvent system. Two bands, wel l resolved from each other , were i so l a t ed and l a b e l l e d as band l a and band lb (Rf va lues , 0.84 and 0.69 r e s p e c t i v e l y ) . In order to confirm that there was no contamination with tocainide i n the i so l a t ed bands, pure tocainide base was also analysed under i d e n t i c a l condit ions as the urine sample and the Rp value was recorded after observation under U . V . l i g h t (Rf = 0 .55) . The crude methanolic extract as wel l as the separated bands, l a and l b , were then examined by high-performance l i q u i d chromatography and gas chromatography/mass spectrometry. - 68 -4. Microbore LCMS of Tocainide Glucuronides A microbore ODS column (5 u h y p e r s i l ODS, 10 cm x 2.1 mm I . D . ) was used for the LCMS analys i s of tocainide g lucuronides . A v a r i a b l e wave length U . V . detector set at 254 nm was placed between the end of the column and the LCMS inter face j e t to obtain a UV trace of the glucuronides p r i o r to the ir i n t r o d u c t i o n into the mass spectrometer. The mobile phase used was a c e t o n i t r i l e : w a t e r (5:95) at a flow rate of 0.2 mL/min and the i n j e c t i o n volume was 2.0 uL. For the LCMS s tud ies , a Hewlett Packard LCMS system (HP 1090 L i q u i d Chromatograph, 5987A LC - GCMS system) was used. The mass spectrometer was used i n the chemical i o n i s a t i o n mode with a source temperature of 2 0 0 ° C . Both pos i t i ve ion and negative ion spectra were obtained for tocainide g lucuronides . 5. I d e n t i f i c a t i o n of Band 1^ as Tocainide Carbamoyl-O-B-D-Glucuronlde 5.1. L i q u i d Chromatographic Analysis of the Hydantoin Derived from the  Glucuronide Toca in ide g lucuronide , corresponding to band 1^ with an Rf value of 0.69 ( i so l a t ed from reverse-phase TLC p l a t e s ) , was d i s so lved i n water and the pH was adjusted to 13 by the add i t ion of 1.0 N sodium hydroxide s o l u t i o n . The mixture was then extracted with 5 mL of dichloromethane. This treatment has been reported by E l v i n et a l . , (1980b) to r e s u l t i n formation of a hydantoin-based s t ruc ture , - 69 -3- (2 ,6-xy ly l ) -5-methylhydanto in . The dichloromethane extract was concentrated on a water bath at 5 5 ° under a stream of n i t rogen . The res idue was d i s so lved i n a mixture of 25% a c e t o n i t r i l e i n 0.05 M potassium chlorate and subjected to chromatographic analys i s using a 5 u ODS column (25 cm x 4.6 mm I .D . ) and 25% a c e t o n i t r i l e i n 0.05 M potassium chlorate as the mobile phase. A standard hydantoin d e r i v a t i v e , supplied by As t ra Pharmaceuticals was a lso subjected to l i q u i d chromatographic analys i s under i d e n t i c a l condit ions to compare the r e ten t ion times. 5.2 GC and GCMS Analysis of the Hydantoin Derived from the Glucuronide The hydantoin d e r i v a t i v e obtained as described above was also analysed by gas chromatography employing SE-30 f u s e d - s i l i c a c a p i l l a r y column (15 m x 0.2 mm) with flame i o n i s a t i o n de tec t ion . The standard hydantoin supplied by Astra Pharmaceuticals was also subjected to chromatograhic ana lys i s under i d e n t i c a l condi t ions to compare the re tent ion times. The same c a p i l l a r y column was also used for the GCMS i d e n t i f i c a t i o n of the hydantoin. Methane was used as the reagent gas to obtain the chemical i o n i s a t i o n mass spectra of the standard hydantoin as w e l l as the hydantoin derived from the glucuronide of tocainide as de sc r ibed . - 70 -5.3 Acid Hydrolysis of the Glucuronide to Tocainide Enantiomers The Band i so l a ted from the TLC plate (R f = 0.69) was d i s so lved i n 1 mL of 1.0 N HC1 i n a 10 mL screw-capped (PTFE l ined) cu l ture tube. The tube was t i g h t l y capped and heated at 1 0 0 ° C for one hour af ter which p e r i o d , the r eac t ion mixture was cooled to room temperature. The excess acid was neut ra l i s ed by the add i t ion of 1.0 N sodium hydroxide s o l u t i o n . An excess of sodium hydroxide was then added and the contents were extracted into 5 mL of dichloromethane. After evaporation of the so lvent , the residue was d i s so lved i n 200 uL of n-hexane and 30 uL of hepta f luorobutyr ic anhydride were added. The t i g h t l y capped tube was heated at 5 5 ° C for 30 minutes. Excess reagent was removed by evaporation with a slow stream of ni trogen at room temperature and the residue was reconst i tuted i n 200 uL of n-hexane. The hepta f luorobutyryl de r iva t ive s of toca in ide , formed by acid hydro lys i s of the glucuronide were analysed on a C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column employing an e l e c t r o n capture detec tor . 5.4 Enzyme Hydrolysis of the Glucuronide to Tocainide Enantiomers The mater ia l (Rf = 0.69) i so l a t ed by preparat ive TLC, was d i s so lved i n 1 mL of acetate buffer (pH = 5.2) and to th i s mixture was added 0.5 mL of P-glucuronidase enzyme (5000 uni t s /mL) . The contents were incubated at 3 7 ° C i n a waterbath for 18 hours. The hydrolysate was made a l k a l i n e by add i t ion of 1.0 N sodium hydroxide so lu t ion and the contents of the tube were extracted into 5 mL of dichloromethane. The - 71 -dichloromethane extract was processed as described before for gas chromatographic a n a l y s i s . 6. Gas Chromatography/Mass Spectrometry (GCMS) of Tocainide  Glucuronide s 6.1 Permethylation of Glucuronides Tocainide glucuronides were permethylated with methyliodide i n dry d imethylsul foxide (DMSO) using methylsul f inylmethide carbanion as c a t a l y s t as per the procedure described by Thompson et a l . , (1973). The procedure, described b r i e f l y , was as fo l lows : 6.1.1 Preparation of Dry DMSO DMSO was d i s t i l l e d fresh before the methylation procedure. DMSO (20 mL) was added to 2.0 g of calcium hydride i n a round bottom f lask and d i s t i l l e d under vacuum at 7 0 ° C u n t i l a 15 mL volume of dry DMSO was c o l l e c t e d . 6.1.2 Preparation of Sodium Methylsulfinylmethide Carbanion  (Dimsylsodium) A 50% d i sper s ion of sodium hydride i n mineral o i l (250 mg, 5 m . moles) was washed three times with anhydrous ether (3 mL) and was q u i c k l y transferred to a round bottom f l a sk containing 5 mL of dry DMSO. The grey coloured suspension was heated gently under n i t rogen - 72 -u n t i l formation of hydrogen ceased ( » 30 minutes) . The r e s u l t i n g straw-coloured so lut ion was stored under nitrogen at - 2 0 ° C u n t i l required for the permethylat ion. 6.1.3 Permethylation To the glucuronide sample i so l a ted by preparat ive TLC and contained i n a 5 mL screw capped v i a l ( R e a c t i - v i a l s ® ) were added 20 uL of DMSO sodium carbanion. The v i a l was connected to a n i t rogen source and after standing for 15 minutes at room temperature, 1.2 uL of methyl iodide were added. The reac t ion was allowed to proceed for one hour at room temperature, af ter which per iod , the reac t ion was stopped by add i t ion of 1 mL of d i s t i l l e d water. The permethylated glucuronide was extracted by shaking with 1 mL of chloroform. The chloroform layer was t ransferred to a clean v i a l and was washed three times with 1 mL of water. The chloroform extract was then concentrated under ni t rogen and stored i n the r e f r i g e r a t o r u n t i l required for analys i s by GC and GCMS. The same procedure was followed for the permethylation of f reeze-dr ied methanol extract of the ur ine and p-Nitrophenol g lucuronide , a pure, commercially a v a i l a b l e , glucuronide as wel l as D-glucuronic a c i d . A Hewlett-Packard 5880 gas chromatograph was used with a model 5987 Hewlett Packard mass spectrometer/HP 1000 mini-computer system. The gas chromatograph was operated i n the s p l i t l e s s i n j e c t i o n mode. The oven was temperature programmed from 5 0 ° to 1 4 0 ° at 3 0 ° C / m i n and from - 73 -1 4 0 ° to 2 4 0 ° at 1 0 ° / m i n . An SE-30 f u s e d - s i l i c a c a p i l l a r y column (25 m x 0.2 mm) and a C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.31 mm) were used for a n a l y s i s . Methane was used as the reagent gas for operat ion i n the chemical i o n i s a t i o n mode. The mass spectrometer was used i n both e l ec t ron impact (EI) and chemical i o n i s a t i o n (CI) mode with a m u l t i p l i e r voltage of 2200 v o l t s , emission current of 300 pA and a source temperature of 2 0 0 ° . - 74 -RESULTS AND DISCUSSION A. Gas-liquid Chromatographic Analysis o f Tocainide Using F u s e d - s i l i c a  C a p i l l a r y Columns The complexity of many b i o l o g i c a l samples requires a n a l y t i c a l methods based on h igh- re so lu t ion chromatography. This requirement i s concerned p r i m a r i l y with the s i tua t ions where multicomponent analyses are needed such as metabolic p r o f i l i n g or screening for numerous drug metabol i te s , but i t i s a lso a v a l i d cons iderat ion i n trace determinations of selected components i n the presence of m e t a b o l i c a l l y un in te re s t ing compounds or contaminants. While column e f f i c i e n c y and the r e s u l t i n g r e so lu t ion are the chief reasons for the increas ing popular i ty of c a p i l l a r y column gas chromatographic techniques, there are other valuable a t t r ibu te s of this methodology. High s e n s i t i v i t y of de tec t ion i s of c r u c i a l importance i n various aspects of drug a n a l y s i s . The sharp peaks, e l u t i n g from a c a p i l l a r y column r e s u l t s i n an increased s i gna l - to -no i se r a t i o over t y p i c a l peaks obtained with packed columns, regardless of the detector used. The noise l e v e l for a c a p i l l a r y system i s genera l ly reduced because the detector performance i s optimised by using appropriate make-up gas at the appropriate flow ra te . This make-up gas flow i s independent of the column flow and the detector s i gna l - to-no i se i s constant. Contaminants from the septum are also reduced because they are constant ly purged. Column bleed i s reduced due to the small amount of the s ta t ionary phase present and c a r r i e r gas flow i s more uniform because f luc tua t ions are dampened by the res i s tance of - 75 -the column. Consequently, peak height i s increased and the noise i s decreased, often r e s u l t i n g i n a 100:1 gain i n the s igna l - to-no i se r a t i o . This increased s e n s i t i v i t y extends the dynamic l i n e a r range of the chromatographic system. The s e n s i t i v i t y gain may be c r u c i a l i n c e r t a i n a p p l i c a t i o n s . Another reason for the increased s e n s i t i v i t y inherent with c a p i l l a r y columns i s due to the i n e r t nature of the s ta t ionary phase and i t s support. The degree of sample adsorption and decomposition occurr ing on the column i s minimized and therefore many l a b i l e substances of pharmaceutical i n t e r e s t can be analysed. This i s e s p e c i a l l y true of f u s e d - s i l i c a c a p i l l a r y columns. Columns prepared from f u s e d - s i l i c a , which i s pure s i l i c o n dioxide (< 1 ppm m e t a l l i c ox ides ) , provide not only exce l l en t chromatographic performance but are f l e x i b l e enough to be nearly unbreakable i n normal usage. The i n e r t nature of the f u s e d - s i l i c a columns and the e f f i c i e n c y of such columns, are assessed by the ana lys i s of a so c a l l e d , " p o l a r i t y mixture" . This mixture includes compounds with a v a r i e t y of funct iona l groups. The column's surface a c t i v i t y and i t s u t i l i t y for a s p e c i f i c separat ion problem can be determined by c a r e f u l l y examining peak shapes of the polar compounds contained i n th i s mixture. Figure 2 shows a chromatogram of p o l a r i t y mixture. The column used was a 50 m x 0.2 mm. I . D . f u s e d - s i l i c a column that had been coated with a 2% so lu t ion of carbowax 20 M. The chromatogram was obtained on a model 5830 A gas chromatograph (Hewlett-Packard) equipped with s p l i t and s p l i t l e s s i n j e c t i o n system and both flame i o n i s a t i o n and e lec t ron capture - 76 -FIGURE 2 C H R O M A T O G R A M O F P O L A R I T Y M I X T U R E O N C A R B O W A X 20M F U S E D S IL ICA C A P I L L A R Y C O L U M N 1. NONANE 2. DECANE 3- UNDECANE A. DODECANE 5- DIBUTYLKETONE 6. TETRADECANE 7. CIS-PROPYLCYCLOHEXANOL 8. TRANS-PROPYLCYCLOHEXANOL 9. 2,6-DIMETHYLAN I LINE 10 20 M I N U T E S 30 Chromatographic Condi t ions: Column, Carbowax 20 M (50 m x 0.2 mm); Injection temperature, 260°C; Detector (F.I.D.) temperature, 260°C; Column temperature, 70°C (5 min) to 220°C at a rate of 10°C/min; Car r ier gas (Helium) flow, 1 ml/min; S p l i t vent flow, 130 ml/min; Inlet pressure, 25 p.s.i ; Make-up gas (Helium) flow, 50 ml/min; Chart speed, 0.3 cm/min. - 77 -de tec tor s . The flame i o n i s a t i o n detector was used i n th i s case and the sample was introduced by the s p l i t mode of i n j e c t i o n . No t a i l i n g was observed for the ketone, a lcohol or the amino compounds contained i n the p o l a r i t y mixture. Base- l ine r e s o l u t i o n was obtained for the two isomeric a l c o h o l s , showing the e f f i c i e n c y as well as the i n e r t nature of the column. Tocainide was f i r s t reacted with hepta f luorobutyr ic anhydride to form a d e r i v a t i v e . The chromatogram obtained by i n j e c t i n g a s o l u t i o n of the heptaf luorobutyrate of toca in ide i n n-hexane at a s p l i t r a t i o of 1:50 i s shown i n f igure 3. A . ^ ^ i e lectron-capture detector was used with argon-methane (95:5) as the make-up gas. The sharp symmetrical peak obtained was considered superior as compared to chromatograms obtained on S i l a r 10 C, OV-225 or SP-2330 glass c a p i l l a r y columns ( f i gure 4, a, b and c ) . The re tent ion time of the peak of toca inide d e r i v a t i v e (7.6 min. at a column temperature of 2 0 0 ° C ) was also considered su i tab le for ana lys i s of toca inide i n a b i o l o g i c a l matrix where a large number of peaks usua l ly appear near the solvent f r o n t . The same carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column was used to optimize condi t ions of a c y l a t i o n of toca in ide with hepta f luorobutyr ic anhydride. a-Bromonaphthalene was chosen as an i n t e r n a l standard i n t h i s study because i t i s unaffected by the presence of HFBA and has good e lec t ron-captur ing proper t i e s . A plot of the area r a t i o s measured at d i f f e r e n t time periods of heating the reac t ion mixture at 5 5 ° C i s shown i n f igure 5. There was a sharp dec l ine i n the area r a t i o s measured for samples which had been heated for 75 minutes or more. However, the - 78 -FIGURE 3 CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVE OF TOCAINIDE ON CARBOWAX 20 M FUSED-SILICA CAPILLARY COLUMN Chromatographic cond i t ions : column, carbowax 20 M f u s e d - s i l i c a c a p i l l a r y (50 m x 0.2 mm). In jec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) flow, 1 mL/min; make-up gas (argon:methane (95:5) f low, 60 mL/min; column i n l e t pressure, 172.3 kPa; chart speed, 0.3 cm/min; s p l i t vent f low, 50 mL/min. - 79 -FIGURE 4 CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVE OF TOCAINIDE Chromatographic cond i t ions : A . Column, s i l a r 10 C glass c a p i l l a r y (10 m x 0.25 mm); i n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature; 2 3 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; make-up gas (argon:methane (95:5) f low, 60 mL/min; column i n l e t pressure, 34.4 kPa; chart speed, 0.3 cm/min; s p l i t vent f low, 40 mL/min. B. Column, 0V-225 glass c a p i l l a r y (50 m x 0.25 mm); oven temperature, 2 3 0 ° C ; column i n l e t pressure 137 kPa. Other condit ions are the same as i n A. C. Column, SP-2330 glass c a p i l l a r y (30 m x 0.25 mm); oven temperature, 2 3 0 ° C ; column i n l e t pressure, 103.4 kPa. Other condi t ions are the same as i n A. - 80 -FIGURE 5 TIME-DEPENDENCE OF DERIVATIVE FORMATION - 81 -area r a t i o s were nearly constant for a l l samples heated from 15 minutes to 60 minutes. Therefore , 30 minutes was chosen as a reasonable time per iod for the durat ion of d e r i v a t i z a t i o n . 1. Selection of a Suitable Internal Standard a-Bromonaphthalene was used as an i n t e r n a l standard during the ear ly development of toca inide i n an ECD assay for rat plasma and u r i n e . Although thi s compound had good e l ec t ron capturing p roper t i e s , i t was not found su i tab le as an i n t e r n a l standard i n the present study because of Its very low re tent ion time. Monoethylg lyc inexyl id ide (MEGX) ( f igure 6 ) , a metabolite of l i d o c a i n e , does form a de r iva t ive with HFBA under the same condit ions as toca inide and has a reasonably long re tent ion time and therefore , the ear ly e l u t i n g endogenous compounds would not i n t e r f e r e . MEGX-HFB also has good chromatographic propert ies and gave a symmetrical peak on the carbowax column. CH 3 H N H — C O — C H 2 — N C 2 H 5 FIGURE 6 - 82 2. Measurement of S p l i t t e r D i f f e r e n t i a t i o n Between Tocainide and  Internal Standard, MEGX A c a p i l l a r y i n l e t s p l i t t e r ' s p l i t s ' the sample into two unequal por t ions , the smaller of which goes onto the column. This reduces the amount of the sample going through the column and prevents column over-l o a d i n g . The major funct ion of the i n l e t s p l i t t e r i s not only to r e s t r i c t the s ize of the sample placed on the column but also to permit the rapid f lu sh ing of the i n j e c t i o n port so that the sample on the column i s followed by pure c a r r i e r gas rather than by exponent ia l ly d i l u t e d sample. S p l i t i n j e c t i o n i s a f l a sh vapor i sa t ion technique, and there ex i s t s a p o s s i b i l i t y of sample d i s c r i m i n a t i o n . D i s c r i m i n a t i o n i s a measure of how wel l the detected peak areas r e f l e c t the o r i g i n a l sample composit ion. Dif ferences i n molecular weight, v o l a t i l i t y , component concentra t ion , p o l a r i t y , s p l i t r a t i o s , in jec ted volume, i n l e t temperature and pressure may a l l a f fect the nature of the s p l i t . Therefore i t was necessary to test for the p o s s i b i l i t y of d i f f e r e n t i a -t i o n of toca in ide and i n t e r n a l standard under d i f f e r e n t s p l i t r a t i o s , keeping a l l other condit ions constant . S p l i t r a t i o s used for th i s study ranged from 1:15 to 1:130. Same i n j e c t i o n volume (1 uL) was used at a l l s p l i t r a t i o s and the area r a t i o s were recorded (Table 1) . These r e su l t s showed that there was no s i g n i f i c a n t d i f f e r e n t i a t i o n between toca inide HFB and MEGX HFB under the condi t ions of the analys i s s ince the area r a t i o s were near ly constant . Table 1 Area r a t i o s at d i f f e r e n t s p l i t r a t i o s s p l i t r a t i o area r a t i o 130 100 75 50 25 15 1.345 1.376 1.286 1.363 1.308 1.357 A s ing le s p l i t r a t i o , optimised for the determination of c a l i b r a t i o n data and p r e c i s i o n were used for the assay of b i o l o g i c a l samples. 3. Minimum Detectable Quantity In order to determine the lower l i m i t of s e n s i t i v i t y for the determination of t o c a i n i d e , picogram quant i t i e s were in jec ted onto the carbowax 20 M c a p i l l a r y column and the s igna l - to-no i se r a t i o was observed. Figure 7 shows the detector response to 3 picograms of toca in ide a r r i v i n g at the detector when a s p l i t r a t i o of 1:30 was used. The volume in jec ted was 1 pL which contained 90 picograms of t o c a i n i d e . 4. A p p l i c a t i o n of C a p i l l a r y gas chromatography f o r the Assay of Rat  Plasma and Urine In order to demonstrate the a p p l i c a b i l i t y of the newly developed c a p i l l a r y column gas chromatographic assay to b i o l o g i c a l samples and to check the r e s u l t s with published data , rat plasma and ur ine samples conta ining toca inide were analysed (Figure 8, A and B ) . Figure 8A shows FIGURE 7 •...'•„ ECD RESPONSE OF 3 PICOGRAMS OF TOCAINIDE Column.carbowax 20 M fused silica capillary(50m.0.2mm) 200*isothermal,split ratio-1:30 a. Chromatographic condi t ions : I n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; column i n l e t pressure, 172.3 kPa, c a r r i e r gas (He) flow ra t e , 1 mL/min; make-up gas (argon:methane, 95:5) 60 mL/min; s p l i t r a t i o , 1:30; chart speed, 0.3 cm/min. FIGURE 8 A. RAT PLASMA PROFILE 3HRS AFTER INTRAVENOUS DOSE OF TOCAINIDE HYDROCHLORIDE B. RAT URINE CONTAINING 1.0ug EACH OF TOCAINIDE AND INTERNAL STANDARD (MEGX) Column: Carbowax 20M (50mx0.25mm) fused silica capillary B 1. TO C A I N ID E 2. MEGX X^jtrtank plasma V blank urine Chromatographic cond i t ions : A . I n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) flow ra te , 1 mL/min; make-up gas (argon:methane, 95 :5) , 60 mL/min; column i n l e t pressure, 172.3 kPa; s p l i t vent f low, 30 mL/min; chart speed, 0.3 cm/min. B. Oven temperature, 1 9 5 ° C . A l l other condit ions are the same as i n A . - 86 -the rat plasma p r o f i l e 3 hours af ter an intravenous dose of toca inide hydrochlor ide along with the ECD trace of blank plasma. No i n t e r f e r i n g peaks were observed i n the plasma and the i n t e r n a l standard (MEGX) was wel l resolved from toca in ide . In the rat urine (Figure 8B) p r o f i l e there was a small peak e l u t i n g very c lose to toca inide which was wel l resolved by reducing the temperature of analys i s to 1 9 5 ° C . C a l i b r a t i o n data and p r e c i s i o n of assay for rat plasma and urine are shown i n Table 2. These data were determined by t r i p l i c a t e analyses of each of s ix concentrations of toca in ide ranging from 50 to 1000 ng along with 1000 ng of the i n t e r n a l standard. The mean r e l a t i v e standard d e v i a t i o n was 8.3% and 6.1% for the plasma and u r i n e , r e s p e c t i v e l y . 5. Pharmacokinetics of Intravenous Tocainide i n the Rat Rat plasma and urine were analysed by the c a p i l l a r y column gas chromatographic method. Plasma data were analysed by a computer program (AUTOAN and NONLIN) and were found to f i t a two compartment model. The pharmocokinetic parameters i n male Wistar rats at three d i f f e r e n t dose l e v e l s of toca in ide are shown i n Table 3, and the cumulative amount of parent drug excreted i n the urine up to a period of 24 hours i s shown i n Table 4. As shown i n Table 3, with increas ing doses, the area under the plasma concentrat ion-t ime curve increased and the t o t a l clearance decreased. Clearance should be constant i f l i n e a r k i n e t i c s apply . This observat ion i s i n agreement with pharmacokinetics of toca in ide i n the rat as reported e a r l i e r (Venkataramanan and Axelson, 1980). These observat ions also ind ica te that a simple l i n e a r pharmacokinetic model i s Table 2 C a l i b r a t i o n curve data and p r e c i s i o n for rat plasma and ur ine assay wt. of toca in ide plasma area ur ine area (ng) r a t i o + S.D. r a t i o + S.D. 50.0 0.0592 + .0083 0.0612 + .0043 100.0 0.1100 + .0144 0.1373 + .0074 200.0 0.2149 + .0020 0.2127 ± .0110 500.0 0.6693 + .0324 0.6376 + .0388 1000.0 1.2781 + 0.1157 1.3358 0.1013 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9989 0.9989 slope = 1.3058 1.3456 in te rcep t = -0.0168 -0.0209 -'88 -Table 3 Pharmacokinetic parameters of intravenous tocainide i n the rats dose : 25 mg/kg 20 mg/kg 15 mg/kg Rat #1 Rat #2 Rat #i3 Rat #4. Rat M§ K ' ( m i n - 1 ) e l 0.0056 0.0058 0.0098 0.0178 0.0071 Co (yg/mL) 8.8805 9.9284 15.5937 19.1727 4.9719 a (min - * ) 0.0252 0.0850 0.1421 0.1264 0.0749 B ( ra in - 1 ) 0.0038 0.0045 0.0048 0.0042 0.0049 A (yg/raL) 5.099 6.407 8.5191 13.2436 3.0882 B (yg/mL) 3.997 2.480 6.9501 4.6972 1.9151 K 1 2 ( m i n - 1 ) 0.0048 0.0249 0.0692 0.0983 0.0221 K 2 1 ( m i n - 1 ) 0.0079 0.0489 0.0512 0.0484 0.0325 T 1 / 2 ( g ) ( m i n ) 183.0 152 143.0 163.0 142.0 AUC (yg .h m L - 1 ) 22.58 23.54 22.11 16.6 10.4 CI (mL/min) 3.98 3.85 3.77 4.45 9.10 V d (mL) 1049.0 858.0 785.0 1061.0 1854.0 - 89 -Table 4 Percent drug excreted unchanged i n 24 h urine Dose 10 mg/kg Dose 20 mg/kg Rat #1 Rat #2 Rat #3 14.2 21.6 29.8 inadequate to descr ibe the k i n e t i c s of toca inide i n the r a t . In the present case only 3 dose l e v e l s were studied i n a l i m i t e d number of r a t s . B . Resolution of Tocainide Enantiomers Tocainide has one c h i r a l center and therefore can ex i s t i n two s tereoisomeric forms ( f igure 9 ) . It i s used c l i n i c a l l y as the racemate which contains equal proportions of both the isomers. It has been shown that the levorota tory isomer i s about 3-fold more potent as an ant iarrhythmic agent than the dextroisomer i n a mouse model (Byrnes et a l . , 1979). Dif ferences i n pharmacological a c t i v i t y between enantiomers are due to t h e i r a b i l i t y to s e l e c t i v e l y in te rac t with asymmetric molecules i n a b i o l o g i c a l system. The most l i k e l y area for such an i n t e r a c t i o n would be the receptor s i t e i t s e l f , but before a drug reaches the receptor s i t e i t undergoes severa l s t e reose lec t ive events (F igure 10). Thus, absorption from the g a s t r o i n t e s t i n a l t r a c t , d i s t r i b u t i o n to var ious body compartments and metabolic pathways can be s t e reose lec t ive ( P a t i l et a l . , 1975; Jenner et a l . , 1980; Low et a l . , 1978). The f i n a l amount of each isomer reaching the receptor s i t e , therefore , depends on - 90 -FIGURE 9 STRUCTURES OF TOCAINIDE ENANTIOMERS R ( — ) T O C A I N I D E H H , C H . C < M - 0 ° H 3 C S ( + ) T O C A I N I D E - 91 -FIGURE 10 STEREOSELECTIVE EVENTS PRIOR TO BIOLOGICAL RESPONSE DRUG DOSE MEMBRANE SELECTIVITY SELECTIVE METABOLISM BIOLOGICAL DRUG RESPONSE RECEPTOR - 92 -the degree of s t e r e o s e l e c t i v i t y of these processes . Pharmacokinetic s tudies of enantiomers can revea l whether the s t e r e o s e l e c t i v i t y l i e s i n the absorption process , d i s t r i b u t i o n , metabolism or a combination of these processes . In order to study the d i s p o s i t i o n of enantiomeric drugs, a very s ens i t i ve and s e l e c t i v e a n a l y t i c a l technique i s required that i s capable of re so lv ing the two isomers when present together i n b i o l o g i c a l f l u i d s . Such a method has been reported for the d i r e c t r e s o l u t i o n of toca inide enantiomers (McErlane and P i l l a i , 1983). This method involves the r e s o l u t i o n of the heptaf luorobutyryl de r iva t ive s of toca in ide on a c a p i l l a r y column coated with an o p t i c a l l y ac t ive s ta t ionary phase, N - i s o b u t y r y l - L - v a l i n e tert-butylamide (Figure 11). C a p i l l a r y columns coated with th i s s ta t ionary phase are commercially a v a i l a b l e under the trade name, C h i r a s i l - V a l ® , and have been demonstrat-ed to be e f f ec t ive for the r e s o l u t i o n of amino acids and amino a lcohols (Frank et a l . , 1980; Solomon and Wright , 1977). In a d d i t i o n , the high thermal s t a b i l i t y of C h i r a s i l - V a l ® made i t po s s ib l e , for the f i r s t t ime, to employ a mass spectrometer coupled to a GLC system for the analys i s of enantiomers ( L i u et a l . , 1981: Frank et a l . , 1978). Figure 12 shows the chromatogram of the hepta f luorobutyryl d e r i v a t i v e of toca in ide enantiomers and of the i n t e r n a l standard extracted from a 24 hour urine sample from a rat dosed with ( ± ) toca in ide hydroch lor ide . Base- l ine r e s o l u t i o n of S(+)tocainide (peak No. 2) and R(- ) toca in ide (peak No. 3) was obtained with a t o t a l chromatographic time of less than 9 minutes. The hepta f luorobutyry l d e r i v a t i v e of monoethylg lyc inexyl id ide ( i n t e r n a l standard) had a reten-- 93 -FIGURE 11 S T R U C T U R E O F T H E C H I R A L S T A T I O N A R Y P H A S E c h i r a s i l - v a l - ;94 -FIGURE 12 ' RAT URINE PROFILE 24HRS AFTER AN INTRAVENOUS DOSE OF (t)TOCAINIDE HYDROCHLORIDE. Column: Chlraall-val glass capillary (25x0.25mm) 1. internal standard 2. S(+)tocainide 3. R(-)tocainide Chromatographic condi t ions : In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 8 0 ° C ; c a r r i e r gas (He) flow ra te , 1 mL/min; s p l i t vent flow, 25 mL/min; make-up gas (argon:methane, 95 :5) , 60 mL/min; column i n l e t pressure, 103.4 kPa; chart speed, 0.3 cm/min. - 95 -t i o n time shorter than that of toca in ide on the c h i r a l c a p i l l a r y column (un l ike t h e i r e l u t i o n pattern from a carbowax 20 M c a p i l l a r y column where re tent ion time of MEGX-HFB was longer than toca inide HFB). This was the f i r s t demonstration of d i r e c t r e s o l u t i o n of toca inide enantio-mers on a c a p i l l a r y column coated with o p t i c a l l y ac t ive s ta t ionary phase. This chromatogram also c l e a r l y showed the d i f f e r e n t i a l d i s p o s i -t i o n of toca in ide enantiomers i n the ra t as evident from the d i f ferences i n peak height of the enantiomers. Equal peak heights were obtained when the racemic drug was analysed (Figure 13). 1. I d e n t i f i c a t i o n of the Resolved Peaks The i d e n t i t y of the peaks was es tabl i shed from a s t e reospec i f i c synthesis of the pure isomers using s t a r t i n g mater ia l s of known o p t i c a l p u r i t y and conf igura t ion (Figure 14). The re tent ion time of the heptaf luorobutyrates of the synthet ic products , t h e i r o p t i c a l r o t a t i o n and the mass spectra were used for i d e n t i f i c a t i o n (Figures 15, 16). Furthermore, the o p t i c a l pur i ty was determined by measuring the area under the two peaks. S tereospec i f i c synthesis of toca inide enantiomers was achieved by the method of Byrnes et a l . , (1979). It consisted of formation of an amide bond between an amino acid and an aromatic amine and i s analogous to procedures es tab l i shed for peptide synthes i s . Carbobenzyloxy D(-) a l a n i n e , a commercially ava i l ab le amine-protected amino acid was allowed to react with 2 ,6-d imethy lan i l ine i n the presence of d i c y c l o h e x y l -carbodi imide . One of the advantages of carbobenzyloxy amino acids i s FIGURE 13 CHROMATOGRAM OF (±) TOCAINIDE HEPTAFLUOROBUTYRATES. Column: Chirasi l-val glass capillary column (25Mx0.25mm) S(+)tocainide 7.9mins R(-)tocainide 8.4mins Chromatographic condi t ions : In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 8 0 ° C ; c a r r i e r gas (He) flow ra te , 1 mL/min; s p l i t vent flow, 25 mL/min; make-up gas (argon:methane, 95 :5 ) , f low, 60 mL/min; chart speed, 0.3 cm/min; column i n l e t pressure, 103.4 kPa. - 97 '--FIGURE 14 SYNTHESIS OF R(-) TOCAINIDE CHjCH COOH NH C O O C H 2 C s H 5 carbobenzyloxy D-alanine .CH 3 + (\ /V-NH2 if* CH 3 2,6-dimethyl aniline dicyclohexylcarbodiimide (DCC) C H 3 V y—NHCOCHNH C O O C H 2 C 6 H 5 \ CH, CH, HBr/HAc U A—NHCOCHNH2- HBr \ _ CH, R(-) Tocainide hydrobromide - 98 -t h e i r general res i s tance to racemization under condi t ions u sua l ly employed for the formation of peptide bonds. In add i t ion to i t s p ro tec t ive e f fect against racemizat ion, the carbpbenzyloxy group possesses further advantage i n the ease with which i t may b'e-<rembved by a number of methods such as c a t a l y t i c hydrogenation at room temperature or the use of hydrogen bromide i n ace t ic acid (Boissonnas, 1963). The product of the r eac t ion between carbobenzyloxy D(-) a lanine and 2 , 6 - d i m e t h y l a n i l i n e , on treatment with a 32% so lu t ion of hydrogen bromide i n ace t i c a c i d , gave a p r e c i p i t a t e of the R(-) toca in ide hydrobromide upon add i t ion of e ther . Ether also d i s so lved the benzylbromide formed during the r e a c t i o n . The product obtained had an enantiomeric composition of 95:5 of the R(-):S(+) isomers, as c a l cu l a ted from the peak areas when t h e i r heptaf luorobutyryl d e r i v a t i v e s were assayed on the C h i r a s i l - V a l ® c a p i l l a r y column. Mass spec t ra l evidence supported the s t ructure of the major isomer. The t o t a l ion current p r o f i l e of the hepta f luorobutyryl d e r i v a t i v e of R(-) toca inide i s shown i n Figure 15 and the e l ec t ron impact mass spectra i n f igure 16. 2. C a l i b r a t i o n Data and P r e c i s i o n of Assay of Tocainide Enantiomers i n Human Plasma and Urine C a l i b r a t i o n curve data and p r e c i s i o n of assay i n plasma are shown i n table 5, and the c a l i b r a t i o n data and p r e c i s i o n of assay i n urine are shown i n table 6. For the plasma assay, peak height r a t i o s rather than area r a t i o s were measured because of an i n t e r f e r i n g endogenous compound e l u t i n g c l o s e l y to the peaks for the toca in ide enantiomers. For the same reason, the plasma assays had to be ca r r i ed out at a s l i g h t l y --99. -FIGURE 15 TOTAL ION CURRENT PROFILE OF HEPTAFLUOROBUTYRYL DERIVATIVE OF R(-) TOCAINIDE 200 zeeee-188G9-16009-14000-12000-10800-8300-eaea-4000-2BB0-' e-468 i i • 608 880 1B0B • ' • • 1288 1400 l I : i • • ' i . . I , l i • ! • • . . r— r~—i r _ i i —r——i i • i 1 r~—i 1 1 1 1 i 8 19 12 14 16 18 28 22 24 26 28 38 TIME (MINS) GC/MS c o n d i t i o n s . Column C h i r a s i l - V a l ® glass c a p i l l a r y (25 m x 0.25 mm) i n j e c t i o n temperature, 2 4 0 ° C ; mode of i n j e c t i o n , s p l i t l e s s ; oven temperature, 5 0 ° C to 1 5 0 ° C at 3 0 ° C / m i n , 1 5 0 ° C to 2 0 0 ° C at 5 ° C / m i n ; source temperature, 2 0 0 ° C ; analyser temperature, 2 4 0 ° C ; t ransfer l i n e temperature, 2 4 0 ° C ; i o n i s a t i o n p o t e n t i a l , 70 ev. - 100 -FIGURE 16 El MASS SPECTRA OF HEPTAFLUOROBUTYRYL DERIVATIVES OF TOCAINIDE ENANTIOMERS ! i ea Sc an 4 I B 1 1 . 2 7 < i n RHTOCAINIDE HFB H,C 14B H , C ' S C / N - \ J > fjC F,C F,COCH N i\ O H,C M 388 5B IBB I5B 286 ^SB 3BB 35B ABB 4SB 58B S5B Sc an 377 13 . 62 m i n . 2<H S(+)TOCAINIDE HFB / C H l 241 H 24 1 CH, O NH COiCFjCFjCF, : i j _ 148 1 « » 169 M 388 I B B 1SB 299 256 3 8 8 3SB 4 8 8 4 5 8 S6B 5SB - 101 -Table 5 C a l i b r a t i o n curve data and p r e c i s i o n of assay of toca inide enantiomers i n human plasma quant i ty of each enantiomer (ng) peak height r a t i o s + S. D. S(+)T0C RSD(%) R(- )T0C RSD(%) 50.0 0.0449 ± 0.0026 5.7 0.0383 ± 0.0075 6.5 125.0 0.1129 ± 0.0136 12.0 0.1048 ± 0.0150 14.3 250.0 0.2181 ± 0.0284 13.0 0.2088 ± 0.0273 13.0 375.0 0.3404 ± 0.0030 0.8 0.3115 ± 0.0074 2.3 500.0 0.4515 ± 0.0136 3.0 0.4110 ± 0.0165 4.0 750.0 0.6748 ± 0.0090 1.3 0.6121 ± 0.0080 1.3 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9998 0.9989 slope = 0.9024 0.8171 in te rcep t = --0.0012 0.0018 The quant i ty of i n t e r n a l standard was 1 ug. - 102 -Table 6 C a l i b r a t i o n data and p r e c i s i o n of assay of toca in ide enantiomers i n human urine quant i ty of each enantiomer (ug) area r a t i o s ± S .D. S(+)T0C RSD(%) R(-•)T0C RSD(%) 0.1 0.1536 ± 0.0080 5.2 0.1630 ± 0.0095 5.8 0.2 0.2532 ± 0.0184 7.2 0.2714 ± 0.0220 8.1 0.4 0.4504 ± 0.0300 6.6 0.4495 ± 0.0300 6.6 0.5 0.5740 ± 0.0402 7.0 0.6102 ± 0.0427 6.9 0.75 0.7954 ± 0.0330 4.1 0.8590 ± 0.0320 3.7 1.00 1.0388 ± 0.0570 5.4 1.1590 ± 0.0080 0.7 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9994 0.9984 slope = 0.9851 1.0982 in te rcept = --0.0596 0.0469 The quanti ty of i n t e r n a l standard was 1 ug. - 103 -d i f f e r e n t column temperature. This adjustment was necessary to resolve the i n t e r f e r i n g peak as far as poss ible from the tocainide peaks so that e r ror s i n quant i t a t ion were minimal . No such i n t e r f e r i n g peaks were observed i n urine or s a l i v a samples (Figures 17A, B & C ) . In order to determine the r e p r o d u c i b i l i t y of area r a t i o s under the optimum condi t ions of the gas chromatographic assay, t r i p l i c a t e i n j e c t i o n s of t r i p l i c a t e samples were made and the r e l a t i v e standard devia t ions were c a l c u l a t e d . Inter sample va r i a t i ons were found to be higher than intra-sample v a r i a t i o n s (Table 7 ) . 3. Preliminary Study of S t e r e o s e l e c t i v i t y i n Tocainide D i s p o s i t i o n i n  Man Two healthy male volunteers par t i c ipa ted i n this study. The chromatographic p r o f i l e s of the ir plasma, ur ine and s a l i v a after an o r a l dose of the racemic drug are shown i n Figure 17 and the plasma concentrat ion-t ime data are given i n Table 8. The plasma concentrat ion measured at each time period showed more rapid disappearance of the R(-) isomer i n both subjects . By 24 hours post-dosing the enantiomer r a t i o i n subject 1 was 1.94 and i n subject 2, 1.49. By 48 hours the r a t io s were 2.53 and 1.49, r e s p e c t i v e l y , i n the two subjects . The plasma samples from subject 2 were a lso analysed by a non-s tereospec i f ic c a p i l l a r y gas chromatographic method employing a carbowax 20 M c a p i l l a r y column which does not resolve the racemate (Table 8 and Table 9 ) . I t can be observed that the to ta l quant i t ie s of S(+) and R(-) enantiomers when assayed i n d i v i d u a l l y using the C h i r a s i l - V a l ® column are i n close agreement with the quanti ty of the racemate employing the carbowax - ioU -FIGURE IT PLASMA, URINE. AND SALIVA PROFILE 24 HOURS AFTER AN ORAL DOSE OF (± )TOCA IN IDE HYDROCHLORIDE 1. internal standard 2S(+) Tocainide 1 3-R(-) Tocainide A 3 JJUL — i — 10 1 0 10 PLASMA URINE SALIVA Chromatographic conditions: A. Column, C h i r a s i l - V a l * glass c a p i l l a r y column (25 m x 0.25 mm); In j e c t i o n temperature, 240°C; detector (ECD) temperature, 350°C; oven temperature, 183°C; c a r r i e r gas (He) flow, 1 mL/min; s p l i t vent flow, 30 mL/min; make-up gas (argon:methane (95:5) flow, 60 mL/min;'column i n l e t pressure, 103.4 kPa; chart speed, 0.3 cm/min. B & C. Oven temperature 180°C. A l l other conditions are same as i n A. - 105 -Table 7 In te r - and intra-sample v a r i a t i o n s i n area r a t i o s S(+)T0C R(-)T0C Sample 1 1. 0.5695 0.6525 2. 0.5512 0.5762 3. 0.5866 0.6121 mean ± SD 0.5691 ± 0.0177 0.6136 ± 0.0381 RSD(%) 3.0 6.2 Sample 2 mean ± SD RSD(%> 1. 0.6503 2. 0.6090 3. 0.6207 0.6266 ± 0.0212 3.3 0.6788 0.6242 0.6503 0.6511 ± 0.0273 4.1 Sample 3 mean ± SD RSD (%) 1. 2. 3. 0.5330 0.5259 0.5206 0.5265 1.1 ± 0.0062 0.5626 0.5907 0.5444 0.5659 4.1 ± 0.0233 mean of 3 samples RSD (%) 0.5740 ± 0.0402 7.0 0.6102 ± 6.9% .0427 - 106 -Table 8 Plasma concentrat ion (ug/mL)-time data fo l lowing an ora l dose of 3 mg/kg toca inide C h i r a l Column Carbowax (20M) Time (h) Subject 1 Subject 2 Subject 2 s(+) R(-) s(+) R(-) T o t a l R,S-0.25 0.585 0.536 0.080 0.083 0.163 0.281 0.50 0.842 0.741 0.287 0.220 0.507 0.565 0.75 0.802 0.728 0.372 0.317 0.689 0.613 1.00 0.793 0.708 0.308 0.248 0.556 0.563 1.50 0.768 0.670 0.425 0.345 0.770 0.897 1.75 0.781 0.696 0.355 0.301 0.656 0.657 2.00 0.844 0.742 0.403 0.352 0.755 0.519 3.00 0.629 0.538 0.368 0.325 0.693 0.545 5.00 0.618 0.508 0.300 0.206 0.506 0.545 7.00 0.481 0.375 0.289 0.252 0.541 0.533 24.00 0.243 0.125 0.176 0.118 0.294 0.270 48.00 0.071 0.028 0.113 0.076 0.189 0.137 72.00 - - 0.049 0.032 0.081 0.086 - 107 -Table 9 C a l i b r a t i o n curve data and p r e c i s i o n of assay of human plasma using carbowax 20 M c a p i l l a r y column Quantity of Tocainide (ng) area r a t i o ± S .D. RSD% 50.0 0.0604 ± 0.0063 10.4 100.0 0.1076 ± 0.0090 8.3 200.0 ' '0.2364 ± 0.0151 6.3 '300.0- 0.3836 ± 0.0110 2.8 500.0 0.6203 ± 0.0282 4.5 1000.0 1.2212 ± 0.0825 6.7 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9994 Slope = 1.2313 Intercept = 0.001 The quant i ty of i n t e r n a l standard was 1 ug . - 108 -column. The h a l f - l i v e s of tocainide enantiomers and of the racemate i n the two volunteers are given i n Table 10. Table 10 H a l f - l i v e s (h) of tocainide enantiomers and the racemate in two healthy volunteers Subject 1 Subject 2 S(+) Tocainide 11.1 25.6 R(-) Tocainide 9.0 20.5 R , S ( ± ) Tocainide 9.2 22.1 The C h i r a s i l - V a l ® glass c a p i l l a r y column (25 m x 0.25 mm) performance deter iorated with repeated analyses of plasma samples. The extent of in ter ference from the c l o s e l y e l u t i n g endogenous peak became more pronounced making quant i ta t ion unre l i ab l e after a time. Therefore , further analyses of plasma, ur ine and s a l i v a were carr ied out by u t i l i z i n g a f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.3 mm) coated with the same s ta t ionary phase, C h i r a s i l - V a l ® . The use of this column resu l ted not only i n better r e s o l u t i o n of the two isomers but also the e l i m i n a t i o n of inter ference from the endogenous substance i n plasma samples (F igure 18). The i n t e r n a l standard used i n th i s case was 1-amino a c e t o x y l i d i d e , W-49167 (Astra Pharmaceuticals) which i s more c l o s e l y r e l a t ed i n s tructure (Figure 19) to tocainide than monoethyl g lyc ine - 109. -FIGURE 18 TOCA IN IDE E N A N T I O M E R S P L A S M A WITH 0.2JUG O F E A C H ENANT IOMER AND 1JJG O F INTERNAL S T A N D A R D ® CHIRASIL-VAL FUSED SILICA CAPILLARY COLUMN 50M>0.3 MM 1. S(+) Tocainide 2. R(-) Tocainide 3. internal standard 0 10 20 30 M I N U T E S Chromatographic condi t ions : In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; make-up gas(N2) flow, 30 mL/min; s p l i t vent flow, 30 mL/min; column i n l e t pressure, 151.7 kPa; chart speed, 0.3 cm/min. FIGURE- 19 STRUCTURES OF 1-AMINOACETOXYLIDIDE (W-49167) AND TOCAINIDE NHCOCHNHo I CHj Tocainide CH, ^ _ V - N H C O C H a N H z CH, In terna l standard - I l l -x y l i d i d e . A primary amine l i k e toca in ide , W-49167 also reacts to produce a monoheptafluorobutyrate de r iva t ive under the condit ions of toca in ide d e r i v a t i s a t i o n (Figure 20) . This i n t e r n a l standard has the a d d i t i o n a l advantage that i t can be used to assay plasma samples even when l i d o c a i n e and i t s metabolites are present (MEGX cannot be used i n such a s i t u a t i o n since i t i s a metabolite of l i d o c a i n e ) . Therefore , a l l further assays were c a r r i e d out by using W-49167 as the i n t e r n a l standard. Table 11 and table 12 show the c a l i b r a t i o n data and p r e c i s i o n of assay for human plasma and urine samples r e s p e c t i v e l y , employing this i n t e r n a l standard. The plasma p r o f i l e 24 hours a f ter admini s t ra t ion of an o r a l dose of the racemate i s shown i n Figure 21. The ur ine p r o f i l e 92 hours a f ter an ora l dose i s shown i n Figure 22. The r e l a t i v e peak heights of S(+) and R(-) enantiomers i n these two chromatograms are i n d i c a t i v e of the extent of s tereospecf ic d i s p o s i t i o n . 4. E f f e c t of Sodium Hydroxide Treatment on Urine Containing  Tocainide-Carbamoyl-O-3-D-Glucuronide I t has been reported (Hasegawa et a l . , 1982) that ester glucuronides are unstable i n a l k a l i n e so lut ions and that they are r e a d i l y hydrolysed, thus generating the parent compound. Since tocainide also forms an ester-type g lucuronide , which i s excreted into the u r i n e , and since the ur ine assay procedure involves add i t ion of sodium hydroxide to l i b e r a t e the free drug, i t was important to e s t a b l i s h that no tocainide was generated from i t s glucuronide during the assay procedure. In order to determine the e f fec t of sodium FIGURE - 20 CI MASS SPECTRA OF HEPTAFLUOROBUTYRYL DERIVATIVE OF INTERNAL STANDARD, W.49167 i ee-i 9B-83-375 ( M t l ) 40-CH, N H C O C H J N H C O C F J C F J C F J C H 3 M.W. 374 i 1 i 4 03 (n»!9) 158 2BB e s B 386 35S 4 86 458 5BB 55B Reagent Gas : Methane - 113 -Table 11 C a l i b r a t i o n curve data and p r e c i s i o n of assay of plasma using C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.31 mm) and the i n t e r n a l standard, W-49167 quant i ty of each enantiomer (ng) area r a t i o s ± S.D. S(+)T0C RSD(%) R(- •)T0C RSD(%) 12.5 0.0366 ± 0.0025 6.8 0.0391 ± 0.0020 5.1 50.0 0.1393 ± 0.0057 4.0 0.1612 ± 0.0080 4.9 100.0 0.2512 ± 0.0250 9.9 0.2729 ± 0.0209 7.6 200.0 0.5634 ± 0.0352 6.2 0.6035 ± 0.0434 7.1 500.0 1.3028 ± 0.0069 0.5 1.3633 ± 0.0410 3.0 1000.0 2.7039 ± 0 . 1 4 5 4 5.3 2.8292 ± 0.1494 5.2 C o r r e l a t i o n c o e f f i c i e n t ; r = 0.9998 0.9998 slope = 2.7089 2.8240 in te rcep t = -0.0036 0.0141 The quant i ty of i n t e r n a l standard was 1 ug. - 114 -Table 12 C a l i b r a t i o n curve data and p r e c i s i o n of assay of ur ine using C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column (50 m x 0.31 mm) and the i n t e r n a l s tandard, W-49167 quant i ty of each enantiomer (ng) area r a t i o s + S.D. R(+)T0C RSD(%) R(- •)T0C RSD(%) 25.0 0.0679 ± 0.0018 2.6 0.0716 ± 0.0041 5.7 50.0 0.1203 ± 0.0050 4.0 0.1253 ± 0.0028 2.2 100.0 0.2306 ± 0.0086 3.7 0.2379 ± 0.0045 1.8 500.0 0.9424 ± 0.0550 5.8 0.9640 ± 0.041 4.2 1000.0 1.8401 ± 0.1335 7.2 1.8735 ± 0.1178 6.2 2000.0 3.6490 ± 0.1020 2.7 3.7264 ± 0.1348 3.6 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9999 0.9999 slope = 1.8074 1.8436 intercept = 0.0346 0.0372 The quanti ty of i n t e r n a l standard was 1 ug. - 115 -FIGURE 21 TOCAINIDE ENANTIOMERS plasma profile 24 hours after oral dose of racemate Li _ 1 1. S(+) Tocainide 2. R(-) Tocainide 3. Internal standard Blank plasma 10 20 M I N U T E S Chromatographic cond i t ions : column, C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y (50 m x 0.3 mm). In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) flow, 1 mL/min; column i n l e t pressure , 151.7 kPa; make-up gas (N 2 ) f low, 30 mL/min; s p l i t vent f low, 30 mL/min; chart speet, 0.3 cm/min. FIGURE 22 TOCAINIDE ENANTIOMERS urine profile 92 hours after oral dose of racemate 1. S(+) Tocainide 2. R(-) Tocainide 3. internal standard — Blank urine ' -' i — — - — i — 0 10 20 M I N U T E S Chromatographic condi t ions : Column, C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y (50 m x 0.3 mm). In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) flow, 1 mL/min; column i n l e t pressure, 151.7 kPa; make-up gas (N2) flow, 30 mL/min; s p l i t vent flow, 30 mL/min; chart speed, 0.3 cm/min. - 117 -hydroxide on production of tocainide i n the u r i n e , an SE-30 f u s e d - s i l i c a c a p i l l a r y column (15 m x 0.25 mm) was used with flame i o n i z a t i o n d e t e c t i o n . Diphenhydramine was chosen as the i n t e r n a l standard, s ince W-49167 was not resolved from tocainide on this phase. Under ivat i sed samples could be analysed on an SE-30 column. Toca in ide , as the free base, eluted from the short column with a re tent ion time of 4 minutes while the i n t e r n a l standard eluted at 2.6 minutes, using a column temperature of 1 6 0 ° C . Both peaks did not exh ib i t any t a i l i n g . The area r a t i o s measured are shown below: pH Area Rat io 7.0 0.053 9.5 0.077 10.0 0.084 13.0 0.075 Since the area r a t io s measured between pH values ranging from 9.5 to 13.0 were not s i g n i f i c a n t l y d i f f e r e n t , i t was assumed that no a d d i t i o n a l amount of tocainide was produced by the act ion of sodium hydroxide on the ester glucuronide (the ester glucuronide undergoes c y c l i z a t i o n , rather than h y d r o l y s i s , to produce a hydantoin d e r i v a t i v e under the inf luence of an a l k a l i ) . At pH 7.0, toca inide (pka 7.8) would be p a r t i a l l y ionized and not e f f i c i e n t l y extracted into the organic - 1 1 8 -l a y e r . Therefore i t was assumed that the use of sodium hydroxide to a l k a l i n i s e urine pr ior to ex t rac t ion with dichlororaethane would not lead to l i b e r a t i o n of tocainide from i t s metabol i tes . C. Pharmacokinetics of Tocainide Enantiomers In Man 1. Pharmacokinetics of Oral Tocainide Enantiomers The plasma concentrat ion-t ime data of each enantiomer, fo l lowing an o r a l dose of 200 mg of ( ± ) tocainide hydrochlor ide tablets to seven healthy male volunteers are shown i n Table 13. Peak plasma l e v e l s [0.850 ± 0.227 yg/mL (n=6) for S(+) isomer and 0.774 ± 0.234 ug/mL (n=6) for R(-) isomer] were found between 1 to 2 hours i n most of the vo lunteer s . However, i n one volunteer , a high plasma l e v e l (1.4 ug/mL of each isomer) was reached wi th in 45 minutes and in another, the peak l e v e l (1.26 ug/mL) was at 3 hours. In the l a t t e r volunteer (SR), plasma concentrat ion remained at high l e v e l (compared to others) for up to 10 hours a f ter the o ra l dose. Plasma l eve l s of each isomer were more or l e s s constant i n a l l the seven volunteers at 24 hours [0.231 ± 0.059 ug/mL of S(+) TOC (n=7) and 0.152 ± 0.058 ug/mL of R(-) TOC (n=7)] and therefore the average r a t i o of the two isomers [(+)T0C/(-)TOC] was 1.52. By 48 hours the plasma concentrations of the two isomers were very low and could not be measured i n most vo lunteers . When i t was measurable, the r a t i o of (+)TOC to (-)TOC var ied from 1.53 to 2.53 with a mean of 1.88 ± 0.56 (n=3). The pharmacokinetic parameters of o r a l toca inide enantiomers are given i n Table 14. The absorption rate constant , K a var ied from 3.708 to 5.616 ( h - 1 ) with a mean of 4.29 ± 0.75 ( h - 1 ) (n=5) for the S(+) isomer and from 3.315 to 4.96 (h~ l ) with a Table 13 Plasma concentration (ug/mL)-tlme data following an oral dose of 200 mg( ±) tocainide hydrochloride In seven healthy volunteers KM JA SR RE CK GK HF Time (hra) (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC 0.25 0.082 0.086 _ _ 0.129 0.143 0.146 0.160 0.476 0.458 - - - -0.50 0.356 0.351 0.355 0.297 0.494 0.494 0.913 0.813 1.180 1.116 0.842 0.774 0.229 0.194 0.75 0.467 0.438 0.472 0.385 0.960 0.933 0.764 0.712 1.402 1.353 0.802 0.728 0.468 0.427 1.00 0.479 0.439 0.494 0.426 0.825 0.776 0.845 0.768 0.998 0.929 0.793 0.708 0.600 0.610 1.25 0.498 0.468 0.535 0.430 - - - - - - 0.786 0.590 - -1.50 0.656 0.622 0.610 0.452 1.105 0.969 - - 1.150 1.078 0.768 0.670 0.813 0.858 1.75 0.758 0.681 0.596 0.485 - - - - - 0.781 0.696 - -2.0 0.697 0.642 0.563 0.435 0.919 0.888 0.888 0.766 1.017 0.912 0.844 0.742 0.588 0.562 3.0 0.762 0.689 0.482 0.418 1.262 1.152 0.758 0.652 0.519 0.431 0.629 0.538 0.493 0.406 5.0 0.653 0.584 0.442 0.369 1.211 1.074 0.632 0.526 0.369 0.315 0.618 0.508 - -7.0 0.563 0.459 0.407 0.315 0.512 0.699 - - 0.344 0.300 0.481 0.375 - -10.0 0.490 0.438 - - 0.738 0.645 0.556 0.426 0.301 0.240 - - 0.308 0.263 24.0 0.333 0.262 0.159 0.085 0.250 0.181 0.235 0.146 0.163 0.109 0.234 0.125 0.231 0.161 30.0 _ _ _ _ - - - 0.173 0.105 - - 0.182* 0.106* 32.0 _ - - - 0.168 0.106 0.152 0.100 - - - - 0.119+ 0.074+ 36.0 _ - - - - - - - 0.160 0.111 - - - -48.0 0.133 0.087 0.063, - 0.051 0.032 0.078 - 0.093 - 0.071 0.028 0.069 -.72.0 0.083 - 0.019 - — - 0.023 — *27 hrs •33 hrs Table 14 Pharmacokinetic parameters of tocainide enantiomers following an oral dose of 200 mg (±) tocainide hydrochloride. KM JA SR RE CK HF GK Mean ± SD K ( h _ 1 ) a (+)T0C (-)TOC 3.708 3.986 4.0934 -4.150 3.315 5.616 4.963 3.907 3.842 4.29 4.08 ± 0.75 ± 0.82 B ( h _ 1 ) (+)T0C (-)TOC 0.034 0.042 0.046 0.076 0.068 0.079 0.047 0.067 0.030 0.048 0.041 0.055 0.046 0.063 0.045 0.061 ± 0.012 ± 0.013 tl/2(B) ( h ) (+)T0C (-)TOC 19.9 16.1 14.9 9.1 10.2 8.7 14.5 10.3 22.8 14.4 16.8 12.6 14.8 12.6 16.3 11.9 ± 4 . 0 ±2.7 AUC (ug.h mL - 1) (+)T0C (-)TOC 22.21 15.91 12.04 7.12 20.48 16.94 17.16 12.03 15.15 10.71 12.73 9.05 15.75 10.33 16.51 11.72 ± 3.76 ± 3.56 - 121 -mean of 4.08 ± 0.82 (h~ ) (n=4) for the R(-) isomer. This corresponded to an absorption h a l f - l i f e of 9.6 minutes and 10 minutes for the S(+) and R(-) isomers, r e s p e c t i v e l y . The d i s p o s i t i o n rate constants , 3 ( h - 1 ) were 0.045 + 0.012 ( h - 1 ) and 0.061 ± 0.013 ( h - 1 ) r e s p e c t i v e l y for the S(+) and R(-) isomers. The h a l f - l i v e s , B(h) were 16.3 ± 4.0 (h) and 11.9 ± 2.7 (h) r e s p e c t i v e l y for the S(+) and R(-) isomers, i n d i c a t i n g t h e i r d i f f e r e n t i a l d i s p o s i t i o n . The log plasma concentrat ion-t ime curves fo l lowing an o r a l dose of the racemate are shown i n Figure 23. Since peak l e v e l s of both the isomers were reached at the same time a f ter o r a l dose and the absorption rate constants , K a , were not s i g n i f i c a n t l y d i f f e r e n t , i t was assumed that the absorption process was not s t e r e o s p e c i f i c . 2. Pharmacokinetics of Intravenous Tocainide Enantiomers The plasma concentrat ion-t ime data fo l lowing an intravenous in fu s ion of 200 m g ( ± ) toca inide hydrochlor ide to f ive healthy male volunteers are given i n Table 15. The i n i t i a l concentrations of the S(+) and R(-) isomers (immediately a f ter the in fus ion was stopped) were 0.748 ± 0.277 ug/mL and 0.714 ± 0.300 ug/mL re spec t ive ly and the mean enantiomer r a t i o was 1.05 ± 0.05. At 24 hours, the mean plasma l e v e l s were 0.180 ± 0.033 pg/mL and 0.105 ± 0.023 ug/mL for the S(+) and R(-) isomers r e s p e c t i v e l y and the enantiomer r a t i o was 1.75 ± 0.35. The enantiomer r a t i o increased to 2.37 ± 0.39 at 48 hours post-intravenous i n f u s i o n . Enantiomer r a t i o s at other times are shown i n Table 16. FIGURE 23 Table 15 Plasma concentration (ug/mL)-time data following an intravenous infusion of 200 mg (±) tocainide hydrochloride to five healthy volunteers KM JA SR RE CK Time :  (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC (+)TOC (-)TOC 0.33 0.635 0.606 0.623 0.551 0.540 0.495 0.708 0.683 1.233 1.237 0.41 0.575 0.538 0.533 0.509 - - - - - -0.50 - - - - 0.542 0.498 - - 1.239 1.272 0.75 0.525 0.467 0.540 0.527 0.526 0.489 0.636 0.593 0.694 0.709 1.0 - - 0.528 0.463 0.515 0.471 0.434 0.399 0.446 0.441 1.5 0.491 0.425 0.480 0.430 0.531 0.487 0.382 0.347 0.416 0.425 2.0 0.413 0.340 0.515 0.463 0.454 0.407 0.372 0.332 0.416 0.421 3.0 0.447 0.387 0.409 0.381 0.463 0.418 0.378 0.322 0.400 0.389 5.0 0.432 0.327 0.438 0.389 0.368 0.322 0.353 0.278 0.393 0.374 7.0 0.375 0.305 0.319 0.282 0.295 0.228 0.287 0.209 0.389 0.367 10.0 - - 0.287 0.233 0.274 0.217 0.268 0.183 0.362 0.364 24.0 0.213 0.134 0.148 0.103 0.162 0.107 0.159 0.069 0.219 0.112 30.0 0.179 0.109 0.129 0.072 0.092 0.057 " 0.121 0.042 0.147 0.074 36.0 0.141 0.072 0.095 0.059 - - - - 0.123* -48.0 0.094 0.043 0.061 0.025 0.041 0.041 0.043 - 0.083 0.040 72.0 0.045 0.018 - - 0.013 — - - — *32.5 hrs Table 16 Enantiomer r a t i o , (+)TOC/(-)TOC, i n the plasma fo l lowing intravenous in fu s ion of 200 mg ( ± ) tocainide hydrochloride to f ive healthy male volunteers Time (h) KM JA SR RE CK Mean ± SD 0.33 1.04 1.13 1.09 1.03 1.00 1.05 ± 0.05 0.75 1.12 1.03 1.07 1.07 0.98 1.05 ± 0.05 1.00 - 1.14 1.09 1.08 1.00 1.07 ± 0.05 1.50 1.16 1.12 1.09 1.10 0.95 1.08 ± 0.07 2.00 1.21 1.11 1.11 1.12 0.99 1.11 ± 0.07 3.00 1.15 1.07 1.11 1.17 1.01 1.10 ± 0.06 5.00 1.29 1.12 1.14 1.26 1.04 1.17 ± 0.10 7.00 1.23 1.13 1.29 1.37 1.03 1.21 ± 0.13 10.0 - 1.23 1.26 1.47 1.14 1.27 ± 0.14 24.0 1.59 1.45 1.51 2.29 1.95 1.75 ± 0.35 30.0 1.64 1.80 1.70 2.90 1.99 2.00 ± 0.51 48.0 2.16 2.45 2.89 - 2.07 2.37 ± 0.39 - 125 -The h a l f - l i v e s ca lcu la ted from the l i n e a r por t ion of the log plasma concentrat ion-t ime curves (Figure 24) for the S(+) and R(-) isomers were 17.05 ± 2.5 hours and 11.74 ± 2.4 hours, r e spec t ive ly (Table 17). A mean di f ference of 5.3 hours was observed between the h a l f - l i v e s of the two enantiomers. The plasma concentrat ion-time data was analysed by a computer program (AUTOAN AND NONLIN) and was found to f i t a two compartment model. The d i s t r i b u t i o n h a l f - l i v e s , t^/2^ a ^ var ied between 0.27 to 1.91 hours (0.81 ± 0.70 hours) for the S(+) isomer and between 0.24 to 2.16 hours (0.86 ± 0.82 hours) for the R(-) isomer. The large v a r i a t i o n of d i s t r i b u t i o n h a l f - l i v e s was due to the comparatively slower d i s t r i b u t i o n i n two vo lunteer s . The intercept of the os-phase (A) also showed wide v a r i a t i o n (ranging from 0.132 to 1.720 Ug/mL) due to the di f ferences i n the i n i t i a l d i s t r i b u t i o n c h a r a c t e r i s t i c s between vo lunteer s . The terminal por t ion of the log plasma concentrat ion-t ime curve was extrapolated to give a ' B ' in tercept which was r e l a t i v e l y constant for a l l subjects (0.456 ± 0.034 ug/mL for S(+) toca inide and 0.448 ± 0.033 ug/mL for R ( - ) t o c a i n i d e ) . Pharmacokinetic parameters ca lcu la ted from the area under the plasma concentrat ion-t ime curves (AUC) of the enantiomers are shown i n Table 18. AUC°° = AUC t + C , . o o t/ 6 Where AUC i s the area under the plasma concentrat ion-t ime curve from o r time 0 to time t (t = time of l a s t concentrat ion measured). C t i s the l a s t concentrat ion measured and 3 i s the slope of the terminal FIGURE 24 TOCAINIDE ENANTIOMERS PLASMA CONCENTRATION-TIME CURVES AFTER INTRAVENOUS INFUSION OF ( ± ) TOCAINIDE 10 20 30 40 50 60 70 TIME(HRS) Table 17 Pharmacokinetic parameters of tocainide enantiomers following an Intravenous infusion of 200 mg (±) tocalnde hydrochloride to five healthy male subjects KM JA SR RE CK Mean ± SD a d T 1 ) (+)TOC (-)TOC 1.4130 1.4038 0.6247 0.5919 0.3610 0.3294 2.4851 2.8632 2.5444 2.8370 1.4856 ± 1.0162 1.6032 ± 1.2060 (+)T0C (-)TOC 0.0333 0.0452 0.0405 0.0610 0.0492 0.0684 0.0446 0.0751 0.0390 0.0551 0.0483 ± 0.0059 0.0609 ± 0.0115 A(yg/mL) (+)TOC (-)TOC 0.1800 0.2332 0.1832 0.1399 0.1319 0.0995 0.355 0.3906 1.7247 1.6389 0.5150 ± 0.6815 0.5000 ± 0.6462 B(ug/mL) (+)TOC (-)TOC 0.4784 0.4231 0.4249 0.4201 0.4491 0.4315 0.4261 0.4938 0.503 0.475 0.4563 ± 0.0339 0.4487 ± 0.0335 K e l ( h _ 1 ) (+)TOC (-)TOC 0.0445 0.0711 0.0569 0.0789 0.0583 0.0890 0.0937 0.1779 0.1613 0.2386 0.0829 ± 0.0474 0.1307 ± 0.0741 ti/2(e) ( h ) (+)TOC (-)TOC 20.8 15.3 17.1 11.4 14.1 10.1 15.5 9.2 17.7 12.7 17.05 ± 2.5 11.74 ± 2.4 t l / 2 ( a ) ( h ) (+)TOC (-)TOC 0.4904 0.4936 1.1093 1.1708 1.9196 2.1629 0.2788 0.2420 0.2723 0.2442 0.8140 ± 0.706 0.8627 + 0.8199 (+)TOC (-)TOC 0.0173 0.0211 0.0365 0.0411 0.0230 0.0256 0.0277 0.0279 0.0640 0.0775 0.0337 ± 0.0183 0.0386 ± 0.0229 k n r ( h _ 1 > (+)TOC (-)TOC 0.0272 0.0500 0.0204 0.0378 0.0353 0.0614 0.0660 0.1500 0.0973 0.1611 0.0492 ± 0.0320 0.0920 ± 0.0586 Table 18 Pharmacokinetic parameters of tocainide enantiomers fo l lowing an intravenous in fus ion of 200 mg ( ± ) tocainide hydrochloride to healthy male subjects KM JA SR RE CK Mean ± SD AUC (ug.h.mL l ) (+)T0C 14.6 10.9 9.7 9.8 13.6 11.7 ± 2.2 (-)TOC 9.4 7.7 6.8 5.5 9.9 7.8 ± 1.8 CLj, (mL/min) (+)T0C 114 152 172 169 115 144.4 ± 28.3 (-)TOC 180 217 245 303 167 222.4 ± 54.5 C1D(mL/min) (+)T0C 44 98 68 52 46 61.6 ± 22.4 (-)TOC 53 113 72 58 54 70.0 ± 25.2 ( V d ) g ( D (+)TOC 204 226 209 228 199 213 ± 13 (-)TOC 235 213 215 242 185 218 ± 22 V c (L) (+)T0C 152 164 172 130 45 132 ± 51 (-)TOC 152 178 188 113 47 135 ± 57 (Vss ( L ) (+)T0C 180 179 174 185 136 171 ± 19 (-)TOC 185 193 193 200 149 184 ± 20 ( V d ) (L/kg) (+)T0C 2.08 2.32 3.14 2.65 2.16 2.47 ± 0.43 (-)TOC 2.40 2.19 3.23 2.81 2.01 2.52 ± 0.49 <Vss ( L / k ^ (+)T0C 1.83 1.84 2.61 2.15 1.48 1.98 ± 0.42 (-)TOC 1.88 1.98 2.90 2.32 1.62 2.14 ± 0.49 - 129 -e l i m i n a t i o n phase. The mean AUC for the S(+) isomer was 11.7 ± 2 . 2 p g . h . m L - 1 and for the R(-) isomer was 7.8 ± 1 . 8 u g . h . m L - 1 . The mean AUC af ter an o r a l dose was much higher [S(+) toca inide = 16.51 ± 3.76 u g . h . m L - 1 ; R(-) toca inide = 11.72 ± 3 . 5 6 p g . h . m L - 1 ] . As a r e s u l t , the b i o a v a i l a b i l i t y ca lcu la ted from the plasma data was 151.7 ± 43.0 percent for the S(+) isomer and 167.5 ± 67.7 percent for the R(-) isomer ( table 19). The b i o a v a i l a b i l i t y of these isomers ca lcu la ted from the CO CO cumulative ur inary excre t ion data [X^ ( o r a l ) / X u ( i . v . ) , was also higher than 100 percent. [S(+) toca inide = 109.7 ± 18.1%; R(-) toca in ide = 112.6] . Table 19 B i o a v a i l a b i l i t y (%) of Tocainide Enantiomers from Plasma Data KM JA SR RE CK Mean ± S.D. (+)T0C (-)TOC) 152.0 169.7 109.8 92.6 211.1 248.7 174.3 218.7 111.4 108.0 151.7 ± 43.0 167.5 ± 67.7 Such high o r a l b i o a v a i l a b i l i t y was observed i n three out of four heal thy male volunteers (100-162%) by another group of inves t i ga tor s but was dismissed as a methodological problem (Lalka et a l . , 1976). There are no other reported values of b i o a v a i l a b i l i t y i n the l i t e r a t u r e with which the present values could be compared. In order to ensure that the unusual ly high o r a l b i o a v a i l a b i l i t y observed i n three out of f ive subjects was not due to an inconsis tency - 130 -i n the dosage forms used (such as degradation of the drug i n the intravenous so lut ion) both the i n j e c t i o n and tablets were assayed by gas chromatography using the C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y column and e l ec t ron capture detec t ion (Table 20). The poss ib le los s of the drug contained i n the intravenous i n j e c t i o n through adsorption by the p l a s t i c tubing used for the in fus ion set ( V e n o c u t ® in fus ion set) was also checked to ensure that the intended dose was administered (Table 21). Since there was no d i f ference i n the area r a t i o s before and a f te r passing the in j ec t ab le so lu t ion through the in fus ion set , i t was assumed that the p l a s t i c tubing does not adsorb any drug during the short contact time employed for the i n f u s i o n . Therefore , the incons i s tency observed i n the area under the plasma concentrat ion-t ime curve a f ter o r a l and intravenous route (AUCpg > AUC^-y) was not due to d i screpanc ies i n the amount of toca in ide i n the dosage form used for the study nor was i t due to loss of the drug during passage through the i n f u s i o n set . Another poss ible reason for the high o r a l b i o a v a i l a b i l i t y of toca in ide may be enterohepatic r e c i r c u l a t i o n as reported for drugs such as indomethacin and sul indac (Kwan et a l . , 1976; Dobrinska et a l . , 1983). While indomethacin (MW = 357) and sulindac (MW = 356) are large molecules and b i l i a r y excret ion has been documented, toca inide i s a r e l a t i v e l y small molecule (MW = 192) and one would not expect the same l e v e l of b i l i a r y e x c r e t i o n . However, tocainide i s known to be metabolized (25-40% of dose) to a g lucuronide , which, because of i t s p o l a r i t y and molecular s ize could undergo b i l i a r y excre t ion . The g lucuronide reaching the g a s t r o i n t e s t i n a l t rac t may be hydrolysed by the g-glucuronidase enzyme ( b a c t e r i a l enzymes, E - c o l i ) and the l i b e r a t e d - 131 -Table 20 Assay of standard toca inide (1.0 yg) and of samples prepared from tablets and i n j e c t i o n s Area Ratios Standard Tablets I n j e c t i o n (+)T0C (-)TOC (+)T0C (-)TOC (+)T0C (-)TOC 1.1499 1.2207 1.1022 1.1340 1.1338 1.2269 1.1022 1.1353 1.0661 1.1491 1.0430 1.1464 0.9517 1.0393 0.9786 1.0611 1.0118 1.0878 X ± SD 1.0701 1.1376 1.0489 1.1147 1.0628 1.1537 ± 0 . 0 8 4 5 ± . 0 9 7 3 ± . 0 6 3 5 ± . 0 4 7 0 ± . 0 6 3 3 ± . 0 6 9 8 % RSD 7.8 8.5 6.0 4.2 5.9 6.0 - 132 -Table 21 Assay re su l t s before and af ter the i n j e c t i o n i s passed through the in fus ion set Area Ratios Before Af te r 1.9245 1.8930 1.8834 1.9045 1.9453 1.9523 X±SD 1.9166 ± .0314 1.9177 ± .0314 % RSD 1.6 1.6 parent drug may be reabsorbed into the systemic c i r c u l a t i o n . This i s e s p e c i a l l y l i k e l y for an unstable ester glucuronide such as that of t o c a i n i d e , s ince i t i s known that deconjugation and reabsorpt ion r e a d i l y take place for drugs such as c l o f i b r i c ac id and d i f l u n i s a l , which also form ester g lucuronides . The process of b i l i a r y r e c y c l i n g would be expected to predominate a f ter o r a l admini s t ra t ion when greater than 90% of the dose passes through the l i v e r , although s i g n i f i c a n t b i l i a r y r e c y c l i n g could also occur a f ter intravenous admin i s t r a t ion . 2.1 Plasma Clearance Plasma c learance , ca l cu la ted by d i v i d i n g the intravenous dose by the area under the plasma concentrat ion-t ime curve for each enantiomer, was 144 ± 28 mL/min (S(+) toca inide) and 222 ± 54 mL/min (R(-) - 133 -t o c a i n i d e ) . (Tota l plasma clearance of the racemate has been reported as 166 ± 14 mL/min (Lalka et a l . , 1976) and 194 ± 33 mL/min (Graffner et a l . , 1980). Pat ients with acute myocardial i n f a r c t i o n had a plasma clearance of 205 ± 64 mL/min (Graffner et a l . , 1980). 2.2 Renal Clearance Renal clearance was ca lcu la ted by the fo l lowing equat ion: X ( i . v . ) C l = ^ AUC ( i . v . ) o OO where X^ i s the cumulative amount of the parent drug i n the urine for a OO period of 96 hours fo l lowing the intravenous i n f u s i o n , and A U C q ( i . v . ) i s the area under the plasma concentrat ion-t ime curve fo l lowing the same dose. Mean renal clearance for the S(+) isomer was 62 ± 22 mL/min and for the R(-) isomer, 70 ± 25 mL/min. (Renal clearance of the racemic drug was reported as 64 ± 6 mL/min by Lalka et a l . , 1976). 2.3 Volume of D i s t r i b u t i o n Volume of d i s t r i b u t i o n , V^ (area) w a s ca lcu la ted as fo l lows : V = Dose ( i . v . ) d(3) 3 AUC ( i . v . ) and the steady state volume of d i s t r i b u t i o n , extrapolated from a s ing le dose study, was ca lcu la ted by: - 134 -where J^t dose given by intravenous in fus ion t durat ion of in fus ion AUMC area under the f i r s t moment curve which i s ca lcu la ted as the t o t a l area under the product of plasma concentra t ion-time vs time, f t . c . dt + O t . c S The mean Vd(3) was 2.47 + 0.43 l i t e r s / k g for S(+) toca in ide and 2.52 ± 0.49 l i t e r s / k g for R(-) toca inide (Apparent volume of d i s t r i b u t i o n of ( ± ) toca inide i n healthy subjects were 1.62 ± 0.22 L / k g , La lka et a l . , 1976; 2.9 ± 0.2 L / k g , Graffner et a l . , 1980). The steady state volumes of d i s t r i b u t i o n were 1.98 ± 0.42 L/kg and 2.14 ± 0.49 L/kg for the S(+) and R(-) isomers r e s p e c t i v e l y . 3. Tocainide Enantiomer Levels i n the Urine The cumulative amount of each isomer excreted unchanged i n the urine fo l lowing an o r a l and intravenous dose of the racemate (200 mg) i g iven i n Table 22. After an o r a l dose, the mean percent excret ion of S(+) isomer was 42.7 ± 14.2 and the R(-) isomer was 32.4 ± 12.5, a d i f f e rence of 10.3% between isomers. The corresponding values a f ter an intravenous in fus ion were 42.4 ± 12.9% (S(+) isomer) and 31.9 ± 13.0% (R(-) i somer) , a d i f ference of 10.5% between isomers. The rena l c o n t r i b u t i o n to e l imina t ion of tocainide was reported to be 40% (La lka et a l . , 1976; Graffner et a l . , 1980. The cumulative amount of t o c a i n i d isomers excreted unchanged i n the urine fol lowing an o r a l dose was higher than that excreted after an intravenous in fu s ion i n four out Table 22 Cumulative excret ion of tocainide enantiomers i n the urine (%) fol lowing a dose of 200 mg ( ± ) tocainide hydroch lor ide . KM JA SR RE CK HF GK Mean ± SD Ora l dose (+)T0C (-)TOC 33.4 22.0 67.7 52.3 52.5 40.9 30.6 19.1 48.3 41.9 27.9 23.5 38.5 27.5 42.7 32.4 ± 14.2 ± 12.5 I . V . Infusion (+)T0C (-)TOC 39.0 29.8 64.2 52.2 39.5 29.5 29.6 15.7 39.7 32.5 42.4 ± 12.9 31.9 ± 13.0 B i o a v a i l a b i l i t y (+)T0C 85.6 105.4 132.9 103.3 121.6 (%) (-)TOC 73.8 100.1 138.6 121.6 128.9 109.7 ± 18.1 112.6 ± 29.9 - 1 3 6 -of f ive healthy vo lunteers . As a r e s u l t , the b i o a v a i l a b i l i t y ca lcu la ted 00 00 from the urine data , X ora l /X i . v . , was higher than 100%. However, u u these values were considerably less than the b i o a v a i l a b i l i t y ca lcu la ted from plasma data . F igure 25 shows the cumulative ur inary excre t ion vs time, and Figures 26 and 27 are representat ive of a log ur inary excret ion rate vs time and log amount remaining to be excreted vs time p l o t s . The data for the excret ion rate vs time p l o t as we l l as the ca l cu la t ions involved are shown i n Table 23. The data for the amount remaining to be excreted vs time p lo t are shown i n Table 24. The h a l f - l i v e s ca lculated from plasma data, rate p lo t and ARE p l o t , fo l lowing both o r a l and intravenous admini s t ra t ion of ( ± ) tocainide hydrochloride to the same subject , are shown i n Table 25. Since the plasma concentrat ion-t ime data of tocainide isomers have been shown to f i t a two compartment model, no CO other pharmacokinetic parameters, other than t ^ ^ a n a " ^ u a r e c a l cu la ted from the ur inary excret ion data. Enantiomer r a t i o s i n the urine fo l lowing an o r a l dose of the racemate are shown i n table 26. In genera l , enantiomer composition of the urine runs p a r a l l e l to that of plasma composit ion. At 24 h , the enantiomer r a t i o , (+) toca in ide / ( - ) toca in ide , i n the urine was 1.41 ± 0.17 (n = 7) while that of plasma was 1.57 ± 0.24 (n = 7 ) . At 48 hours the r a t i o s were 2.03 ± 0.53 (n = 7) i n the urine and 1.81 ± 0.46 (n = 11) i n the plasma. The ur inary excre t ion rate constant , k e , was determined as fo l lows : k e = K E x f where ' f i s the f r a c t i o n of the drug excreted unchanged i n the urine - £37'*-FIGURE 25 CUMULATIVE AMOUNT OF SWTOCAINIDE EXCRETED IN THE URINE 20 40 60 80 TIME(h) S U B J E C T : CK - 1 3 8 -FIGURE.26 SEMILOG ARITHMIC PLOT OF EXCRETION RATE VERSUS TIME AFTER INTRAVENOUS ADMINISTRATION OF (OTOCAINIDE HYDROCHLORIDE 4000, r 1000 " t t I S(+) tocainide T - 0.9428 6 » 0.0391 hr' tlj = 17.7 hrs •1 O—o R ( - ) tocainide T c 0.9518 g » 0.0463 hr tH • 14.9 hrs 1 U X 100 101 20 40 60 TIME(h) 80 S U B J E C T : CK - 139 -FIGURE 27 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 A F T E R I N T R A V E N O U S A D M I N I S T R A T I O N O F T O C A I N I D E H Y D R O C H L O R I D E 40 10 cn £ 3 X i 8 3 X 10 o \ « % • — • S(+) tocainide r » 0.9948 B « 0.0481 h"1 t>s - 14.4 hrs O—O R ( - ) tocainide r = 0.99S4 8 " 0.0601 h"1 th - 11.5 hrs •11 . , , _ 20 40 60 80 T I M E(h) SUBJECT: CK Table 23 Urinary excretion data for the rate p l o t . Dose: l . v . Infusion of 200 mg (±) tocainide hydrochloride at a rate of 10 mg/mln. pl o t t i n g t o t a l t o t a l excretion excretion c o l l e c t i o n time (h) volume (+)T0C (-)TOC rate (+)T0C rate (-)TOC time (h) (mL) PH ( p g ) ( U g ) (Pg/h) (Pg/h) 1.0 0.5 98.0 6.8 2886 3010 2886 3010 2.0 1.5 46.0 5.6 1989 2074 1989 2074 3.0 2.5 73.0 5.5 2638 2548 2638 2548 5.0 4.0 540.0 6.5 3068 2913 1534 1456 7.0 6.0 473.0 6.0 3802 3424 1901 1712 9.0 8.0 296.0 6.3 2371 2242 1186 1121 11.0 10.0 210.0 6.8 1374 1108 687 554 12.5 11.7 386.0 7.3 824 668 549 445 13.5 13.0 102.0 6.6 421 337 421 337 19.0 16.2 445.0 5.8 4860 4249 884 772 i 21.0 20.0 74.0 5.3 1350 996 675 498 -24.0 22.5 60.0 5.9 763 599 254 199 S 27.0 25.5 446.0 5.5 1772 1170 591 390 i 28.0 27.5 514.0 6.3 635 241 635 241 30.0 29.0 126.0 5.5 446 277 223 138 32.5 31.2 56.0 5.2 933 668 373 267 37.0 34.7 130.0 5.3 1399 870 311 193 43.0 40.0 296.0 5.6 2995 2450 499 408 46.0 44.5 122.0 5.4 1432 853 477 284 53.0 49.5 338.0 6.8 774 386 111 55 58.0 55.5 406.0 7.0 377 123 76 25 63.0 61.5 192.0 6.2 588 323 118 65 68.0 65.5 360.0 6.0 669 294 134 59 72.0 70.0 150.0 6.0 414 235 103 59 78.0 75.0 164.0 5.2 122 49 31 8 82.0 80.0 160.0 5.2 303 120 75 30 86.0 84.0 240.0 5.3 178 72 44 18 96.0 91.0 410.0 5.2 325 179 32 17 - 141 -Table 24 Urine data for ARE p lot of S(+) toca inide Dose: IV in fus ion of 200 mg ( ± ) toca inide hydrochlor ide at a rate of 10 mg/min. Amount remaining Time amount excreted cumulative amount to be excreted (hrs) (mg) (mg) (mg) 0- 0 - - 40.70 (X~) 0- 1 2.886 2.886 37.81 1- 2 1.989 4.875 35.82 2- 3 2.638 7.513 33.18 3- 5 3.068 10.581 30.12 5- 7 3.802 14.383 26.31 7- 9 3.802 16.754 23.94 9-11 1.374 18.128 22.57 11-12.5 0.421 20.373 20.32 12.5-13.5 4.360 25.233 15.46 13.5-19 4.860 25.233 15.46 19-21 1.350 26.583 14.11 21-24 0.763 27.346 13.35 24-27 1.772 . 29.118 11.58 27-28 0.635 29.753 10.94 28-30 0.446 30.199 10.50 30-32.5 0.933 31.132 9.56 32.5-37 1.399 32.531 8.16 37-43 2.995 35.526 5.17 43-46 1.432 36.958 3.74 46-53 0.774 37.732 2.96 53-58 0.377 38.109 2.59 58-63 0.588 38.697 2.00 63-68 0.669 39.366 1.33 68-72 0.414 39.780 0.92 72-78 0.122 39.902 0.79 78-82 0.303 40.205 0.49 82-86 0.178 40.383 0.31 86-96 0.325 40.708 0.00 Table 25 H a l f - l i f e (hours) of tocainide enantiomers calculated from plasma and urine data K M JA SR RE CK HF GX Mean * SD Plasma .oral (+)TOC (-)TOC •i.v. (+)TOC (-)TOC 19.9 16.1 20.8 15.3 14.9 9.1 17.1 11.4 10.2 8.7 14.1 10.1 14.5 10.3 15.5 9.2 22.8 14.4 17.7 12.7 16.8 12.6 14.8 12.6 16.3 ± 4.0 11.9 ± 2.7 17.0 ± 2.5 11.7 ± 2.4 Urine Rate Plot i_oral • i.v. (+)T0C (-)TOC (+)T0C (-)TOC 14.1 11.4 19.1 15.6 11.3 8.5 11.6 10.0 13.4 10.9 16, 12. 18.6 13.5 20.6 12.3 15.6 13.7 17.7 14.9 14.6 ± 2.7 11.6 ± 2.1 17.1 ± 3.4 13.0 ± 2.2 ARE Plot i—oral l - i . v . (+)TOC (-)TOC (+)TOC (-)TOC 10.8 9.6 15.9 13.8 7.5 6.1 10.7 11.5 9.7 13.7 8.6 13.2 10.6 16.3 11.9 11.1 12.1 14.4 11 .5 10.8 ± 2.0 9.6 ± 2.2 14.2 ± 2.2 11.4 ± 1.8 Table 26 Enantiomer r a t i o , (+)T0C/(-)TOC, in the plasma and urine following an oral dose of 200 mg ( t ) tocainide hydrochloride to 7 healthy male volunteers. KM JA SR RE CK GK HF i L IUC (h) P* U* P U P U P u P U P U P U 0.25 0.95 - - - 0.90 - 0.88 - 1.04 - - - - -0.50 1.01 - 1.19 - 0.91 - 1.12 - 1.06 - 1.09 - 1.18 -1.00 1.09 1.04 1.16 1.02 1.06 1.02 1.10 1.05 1.07 1.01 1.10 1.03 0.98 0.92 2.0 1.08 1.04 1.29 1.04 1.03 1.16 1.16 1.09 1.11 0.98 1.14 1.12 1.04 0.94 3.0 1.10 1.13 1.15 0.99 1.09 1.04 1.16 1.18 1.20 1.58 1,17 1.15 1.21 0.95 5.0 1.12 1.19 1.20 1.15 1.13 1.05 1.20 1.26 1.17 1.09 1.22 1.15 - 0.96 7.0 1.23 1.31 1.29 1.22 1.16 1.18 - 1.21 1.15 1.12 1 .28 1.17 - 0.96 10.0 1.12 - - 1.22 1.14 1.19 1.30 1.29 1.25 1.20 - - 1.17 1.32 24.0 1.27 1.48 1.87 1.36 1.38 1.40 1.71 1.72 1.50 1.30 1.87 1.45 1.43 1.16 48.0 1.53 2.60 - 1.82 1.59 1.73 - 2.75 - 1.61 2.50 2.36 1.62 1.39 72.0 - 2.97 - 2.28 - 2.00 - 3.09 - 1.50 - 3.79 - 1.50 *P " plasma U = urine - 144 -(which i s determined from % X ) and K „ i s the o v e r a l l e l imina t ion rate u ti constant . The mean ur inary excret ion rate constant , k e for the S(+) toca in ide was 0.0337 ± 0183 h - 1 and for the R(-) isomer 0.0386 ± 0.0229 h - 1 . The non-renal e l i m i n a t i o n rate constant , k n r , i n c l u d i n g metabolic e l i m i n a t i o n , i s given by: k n r = K E (1 - f ) . There was wide v a r i a t i o n i n the values of k^- i n d i f f e r e n t subjects (0.0492 ± 0.32 h " 1 for S(+) t o c a i n i d e , 0.0920 + 0.0586 h ~ 1 for R(-) isomer) presumably because of the d i f ferences i n metabolism. The high values of non-renal e l i m i n a t i o n rate constant for the R(-) isomer was expected because of i t s fa s ter metabolic e l i m i n a t i o n (g lucuronidat ion?) ( t ab le 17). The d i f ferences i n pharmacokinetic parameters such as t ]_/2» Cl.p, AUC, k ^ between the two enantiomers of toca in ide i n heal thy volunteers c l e a r l y points to a s t e reose lec t ive d i s p o s i t i o n . Dif ferences i n s t e r e o s e l e c t i v i t y may be due to d i f ferences i n the rate of absorpt ion , d i s t r i b u t i o n , metabolism or excre t ion . Since no d i f ference was observed i n the absorpt ion, d i s t r i b u t i o n and excre t ion , i t i s assumed that the s t e r e o s e l e c t i v i t y i s i n the proces's-of metabolism. Since toca inide i s known to be metabolized to a glucuronide and s ince the g lucuronida t ion process i s known to be s t e reose lec t ive for many c h i r a l drugs, i t i s most l i k e l y that g lucuronidat ion i s the s tereo-s e l e c t i v e step involved i n the d i f f e r e n t i a l d i s p o s i t i o n of toca in ide isomers. Figures 28a and 28b show the r e l a t i v e proportions of both -~145 -FIGURE 28-' URINE PROF ILE B E F O R E ( A ) AND A F T E R ( B ) H Y D R O L Y S I S WITH ^ -GLUCURON IDASE 1 . S ( + ) t o c a l n i d e 2 . R ( - ) t o c a i n i d e 3. I n t e r n a l s t a n d a r d Chromatographic condi t ions : Column, C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y (50 m x 0.3 mm); i n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; make-up gas (N2) f low, 30 mL/min; s p l i t vent f low, 30 mL/min; column i n l e t pressure , 151.7 kPa; chart speed, 0.3 cm/min. - 146 -isomers i n the same urine sample before and after hydro ly s i s with 3-glucuronidase enzyme (Source: bov ine l i v e r ) • The amountof R(-) isomer generated after hydro lys i s was higher than that of the S(+) isomer showing that the R(-) toca inide glucuronide was i n excess i n the u r i n e . 4. Chromatographic Analysis of Uremic Plasma Extract on C h i r a s i l - V a l 8  F u s e d - s i l i c a C a p i l l a r y Column (50 m x 0.31 mm) Figure 29 i s the blank uremic plasma p r o f i l e showing the large number of peaks due to endogenous compounds present i n the plasma. One of the endogenous compounds e l u t i n g at 8.7 minutes did i n t e r f e r e with-'the ana lys i s of R(-) isomer which had an i d e n t i c a l re tent ion time ( F i g . 29) . Changing the chromatographic condit ions such as column temperat-ure , c a r r i e r gas (He) flow r a t e , use of the H2 as c a r r i e r gas, or temperature-programming did not improve the r e s o l u t i o n of t h i s endo-genous compound from the R(-) toca inide isomer and therefore i t was considered impossible to use t h i s c a p i l l a r y column for the assay of uremic plasma samples. At t h i s time i t was decided to t ry a combination of columns of d i f f e r e n t p o l a r i t y to resolve the endogenous component i n the plasma from tocainide isomers. It was known from our own exper i -ments with a carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column that serum samples containing c rea t in ine l e v e l s as high as 16.8 mg% did not show any extra peaks i n the chromatogram that would i n t e r f e r e with toca inide determinations (Figure 30). Six serum samples containing c rea t in ine i n the range of 4.9 to 16.8 mg%, obtained from Vancouver General H o s p i t a l , were extrac ted , d e r i v a t i s e d and analysed on a carbowax 20 M c a p i l l a r y - 147 -FIGURE 29 GC/ECD PROFILE OF UREMIC PLASMA BLANK ON CHIRASIL-VAL© FUSED-SILICA CAPILLARY COLUMN kJL__ Chromatographic cond i t ions : In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 7 0 ° C (13 min) to 1 9 0 ° C at 5 ° C / m i n ; c a r r i e r gas (H2) f low, 1 mL/min; make-up gas (N2) f low, 30 mL/min; s p l i t vent f low, 30 mL/min; column i n l e t pressure, 151.7 kPa; chart speed, 0.3 cm/min. - 148 -FIGURE 30 CHROMATOGRAM OF BLANK UREMIC PLASMA EXTRACT CONTAINING 16.8 MG% CREATININE ON CARBOWAX 20 M FUSED-SILICA CAPILLARY COLUMN Chromatographic cond i t ions : Column, carbowax 20 M f u s e d - s i l i c a c a p i l l a r y (50 m x 0.2 mm). In j ec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; make-up gas (argon: methane, 95 :5 ) , 60 mL/min; column pressure, 1 9 0 ° C ; s p l i t vent flow, 30 mL/min; chart speed, 0.3 cm/sec. (50 m x 0.2 mm) - 149 -column. None of these samples exhibi ted i n t e r f e r i n g peaks. Therefore a a carbowax 20 M f u s e d - s i l i c a c ap i l a ry column (20 m x 0.2 mm) was connected in ser ies with a C h i r a s i l - V a l ® column (50 m x 0.31 mm). The r e s u l t i n g chromatogram i s shown i n Figure 31. The chromatogram of the blank uremic plasma did not contain any extra peaks that would i n t e r f e r e with tocainide determinat ion. A very clean chromatogram, with exce l l ent r e s o l u t i o n of the two enantiomers, was obtained for the plasma c o l l e c t e d from a pat ient with rena l dysfunc-t i o n . Therefore , for a l l further analyses , the two columns connected i n ser ies were used (Figure 31) . C a l i b r a t i o n data and p r e c i s i o n of plasma assay using the dual c a p i l l a r y column i s shown i n Table 27. 5. Pharmacokinetics of Tocainide Enantiomers i n Renal Dysfunction and  During Hemodialysis In view of the extent of ur inary excret ion of tocainide (40% of dose as i n t a c t drug i n normal subjects and 20-70% i n disease states) and i t s major, metabol i te , tocainide carbamoyl-O-0-D-glucuronide (TOCG) (25-40% of dose) , the e f fec t of rena l i n s u f f i c i e n c y on e l i m i n a t i o n of these substances should be quanti tated to permit r a t i o n a l dosing adjust-ment. Besides the r i s k of accumulation of drugs and toxic metabolites i n renal i n s u f f i c i e n c y , c e r t a i n p h y s i o l o g i c a l and anatomical changes a lso a l t e r the pharmacokinetic parameters of drugs. For example, the volume of d i s t r i b u t i o n of d igoxin i s decreased due to changes i n d i s t r i b u t i o n c h a r a c t e r i s t i c s ( G i b a l d i and P e r r i e r , 1972; Reuning et a l . , 1973). M o d i f i c a t i o n of erythrocyte concentrations can a lso d i s turb the volume of d i s t r i b u t i o n of some drugs. Anemia i s accompanied by a - 150 -FIGURE 31 GC/ECD PROFILE OF UREMIC PLASMA 2 HOURS AFTER INTRAVENOUS INFUSION OF ( ± ) TOCAINIDE TO AN ANEPHRIC PATIENT A. CHIRASIL-VAL® FUSED-SILICA CAPILLARY COLUMN B. CARBOWAX 20 M AND CHIRASIL-VAL® COCO B i O C N Chromatographic c o n d i t i o n s : A. In jec t ion temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 7 0 ° C (13 min) to . 1 9 0 ° C at 5 ° C / m i n ; c a r r i e r gas (H2) f low, 1 mL/min; make-up gas (N2) f low, 30 mL/min; s p l i t vent f low, 30 mL/min; column i n l e t pressure, 151.7 kPa; chart speed, 0.3 cm/min. B. Oven temperature 2 0 0 ° C ; column i n l e t pressure, 179.3 kPa . Other condi t ions are same as i n A. - 151 -Table 27 C a l i b r a t i o n data and p r e c i s i o n of uremic plasma assay using dual c a p i l l a r y columns (carbowax 20 M (20 x 0.2 mm) and C h i r a s i l - V a l ® (50 m x 0.31 mm). quant i ty of each enantiomer (ng) area r a t i o S(+)T0C RSD(%) R(- •)T0C RSD(%) 50.0 0.1204 ± 0.0051 4.2 0.1217 ± 0 . 0 0 6 8 5.6 100.0 0.2111 ± .0044 2.1 0.2134 ± 0.0070 3.3 200.0 0.3832 ± .0173 4.5 0.4002 ± 0.0186 4.6 500.0 0.9700 ± 0.0443 4.5 1.0118 ± 0.0480 4.7 1000.0 2.1040 ± .1264 6.0 2.1077 ± 0.1131 5.3 mean RSD ± SD 4.2 ± 1.4 4. 7 ± 0.8 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9988 0.9996 slope = 2.0889 2.0951 in te rcep t = -0.0151 -0.0042 The quant i ty of i n t e r n a l standard was 1 ug. - 152 -decreased binding of gentamicin to red blood c e l l s and consequently plasma concentrations become much higher than i n pat ients with normal hemoglobin l eve l s (R i f f and Jackson, 1971). Funct iona l and anatomical changes caused by chronic uremia may a f f ec t , to some degree, the absorp-t i o n of drugs given o r a l l y ( P e t i t p i e r r e , 1972; Fabre et a l . , 1967). In a d d i t i o n to these changes, renal i n s u f f i c i e n c y occa s iona l ly causes a l t e r a t i o n s to the metabolism of drugs. In uremic pat ients treated with phenytoin, serum concentrations of t o t a l drug are lower than i n normal subjects and the plasma h a l f - l i f e i s decreased. This unusual phenomenon re su l t s from an increased transformation rate of phenytoin to i t s p r i n c i p a l metaboli te (5-phenyl-5-p-OH-phenyl hydantoin) (Melk et a l . , 1970; Odar-Cederlof and Borga, 1974). E l iminated p r i m a r i l y i n the u r i n e , t h i s metabolite a t ta ins r e l a t i v e l y high serum l e v e l s i n uremia. In other cases, renal disease decreases the f ormation of the metabol-i t e s . For example, uremia decreases a c e t y l a t i o n of su l f i soxazo le and p - a m i n o s a l i c y l i c acid (Reidenburg et a l . , 1969; Ogg et a l . , 1968). 5.1 Pharmacokinetic of Intravenous Tocainide Enantiomers Plasma concentrat ion-t ime data fo l lowing an intravenous i n f u s i o n of 200 mg of ( ± ) toca inide hydrochlor ide to a pat ient with renal dysfunct ion are shown i n table 28. The subject was a 22 year o ld male who had been undergoing d i a l y s i s three times a week for two years (serum c r e a t i n i n e concentra t ion , 13.4 mg%). Blood samples were c o l l e c t e d up to 24 hours fo l lowing drug i n f u s i o n , at the 25th hour, hemodialysis was s tar ted and the d i a l y s i s and blood sampling continued for 5 hours . Even though the plasma l eve l s were lower than i n healthy i n d i v i d u a l s who have -"153 -Table 28 Plasma concentration-time data fo l lowing intravenous in fus ion of 200 mg ( ± ) toca inide hydrochloride to a pat ient with renal dys funct ion . Time (+)T0C (-)TOC r a t i o (h) (yg/mL) (ug/mL) (+)T0C/(-)T0C 0.33 0.432 0.418 1.03 0.50 0.329 0.316 1.04 0.75 0.329 0.321 1.02 2.00 0.333 0.314 1.06 3.00 0.346 0.326 1.06 5.0 0.266 0.230 1.15 10.0 0.261 0.228 1.14 12.0 0.244 0.198 1.23 24.0 0.2021 0.149 1.35 - 154 -received the same dose, the plasma concentrat ions pers i s ted i n renal f a i l u r e , i n d i c a t i n g a p o s s i b i l i t y of accumulation of tocainide to tox i c l e v e l s on repeated dosing to pat ients with kidney d i sease . The h a l f - l i f e ca lcu la ted from the l i n e a r port ion of the log plasma concentrat ion time curve (Figure 32) was 45.3 hours for the S(+) isomer and 28.7 hours for the R(-) isomer (Table 29) . These values correspond to 2.5 fo ld increase i n h a l f - l i f e as compared to normal sub ject s . Wiegers et a l . , (1983) have reported h a l f - l i v e s ranging from 16.6 to 42.7 hours for the racemate in a group of 9 pat ients with t o t a l renal f a i l u r e ( c r e a t i n i n e clearance < 5 mL/min). Oltmans et a l . , (1983) also observed a h a l f - l i f e of 57.4 hours for the racemate i n a pat ient with act ive hepatic necros i s and rena l dys funct ion . Plasma clearance was 86 mL/min for the S(+) isomer and 146 mL/min for the R(-) isomer which corresponds to a reduct ion of 40% from normal subjects . This i s cons i s tent with the observat ion that the rena l c o n t r i b u t i o n to t o t a l clearance i s approximately 40% i n healthy sub jec t s . The volume of d i s t r i b u t i o n ( V a ) i s s i g n i f i c a n t l y increased compared to the i n healthy sub ject s . The (Vj) 8 of the S(+) isomer was 338 l i t e r s and that of R(-) isomer was 365 l i t e r s . During a 5 hour hemodia lys i s , the h a l f - l i f e was 6.5 hours for the S(+) isomer and 5.4 hours for the R(-) isomer. Wieger et a l . (1983) also have observed a h a l f - l i f e of 8.5 ± 4.6 hours for the racemate in a group of 9 pat ients with renal dys funct ion . The change i n enantiomer composition with time was s i m i l a r to the observation i n healthy subjects . At 24 hours post i n f u s i o n , the enantiomer r a t i o , (+) T0C/(-) TOC, was 1.35. - • 155^ -FIGURE 32 LOQ PLASMA CONCENTRATION-TIME CURVE FOLLOWING INTRAVENOUS INFUSION OF 200 MQ OF (t)TOCAINIDE HYDROCHLORIDE TO A PATIENT WITH RENAL DYSFUNCTION. E a a UJ o z o u < 2 CO < 10 15 TIME (HRS) S ( + ) t o c a i n i d e r=0.9810 ^ = 0 . 0 1 5 3 h r " t^=45.3 h r s R ( - ) t o c a i n i d e r=0.9691 0.0241 h r " .75 h r s 20 25 Table 29 Pharmacokinetic parameters of tocainide enantiomers fol lowing an intravenous in fus ion of 200 mg ( ± ) tocainide hydrochloride to a patient with renal dysfunction and during 5 hr hemodia lys i s . Patient Healthy subjects (n=5) (+)T0C) (-)TOC) (+)T0C) (-)TOC) AUC (ug.h.raL l ) 19.29 11.35 11.7 + 2.2 7 .8 + 1 .8 t l / 2 ( g ) ( h r s ) 45.3 28.7 17.05 + 2.5 11.7 + 2.4 C 1 T (mL/min) 86.0 146.0 144.0 + 28 222.0 + 54 ( V d ) p (L) 338.0 365.0 213.0 + 13 218.0 + 22 ( V d ) g (L/kg) 4.50 4.86 2.47 + 0.43 2.52 + 0.49 C1 D (mL/min) 20.0 ± 4 . 8 49.8 ± 16.3 - -t l / 2 ^ n r s ^ during d i a l y s i s 6.5 5.4 - -f r a c t i o n removed by d i a l y s i s 0.353 0.385 - -% removed by d i a l y s i s 35.0 38.0 - -- 157 -5.2 D i s p o s i t i o n of Drug Glucuronides i n Renal F a i l u r e Glucuronide conjugation has not been extens ive ly inves t iga ted i n pat ients with renal f a i l u r e . Studies i n rats have shown that the g lucuron ida t ion process may be diminished (p-aminobenzoic a c i d ) , increased (naphthol) or unaffected (4-methylumbelliferone) i n e x p e r i -mental rena l f a i l u r e (Canalese et a l . , 1980; Howie and Bourke, 1979; Leber et a l . , 1972). Recent reports ind ica te regenerat ion of parent drug from c e r t a i n glucuronide conjugates i n rena l f a i l u r e (Faed and McQueen, 1979; Gugler , 1979a; Gugler et a l . , 1979b). Although excre t ion of g lucuronides by the kidneys in to the urine re su l t s i n the i r r e v e r -s i b l e e l i m i n a t i o n of the conjugates from the body, t h i s i s not n e c e s s a r i l y true of the glucuronides excreted into the b i l e . Fol lowing b i l i a r y e x c r e t i o n , glucuronides may become substrates for the micro-f l o r a l ^-glucuronidase of the g a s t r o i n t e s t i n a l t r a c t , permit t ing reabsorptdLon of the l i b e r a t e d parent compound (Okol ic sanyi et a l . , 1971; H a r t i a l a , 1973). Hydrolys i s of the glucuronides can also be c a r r i e d out by the lysosomal (3-glucuronidase which i s present i n most t i s sue s ; p a r t i c u l a r l y l i v e r , k idney, spleen and i n t e s t i n a l epi thel ium (Levy and Couchie, 1966; Wakabayashi, 1970). F a i l u r e to excrete a g lucuronide may lead to hydro ly s i s of the conjugate, leading to accumulation of the ac t ive compound i n renal f a i l u r e . An example i s provided by c l o f i b r i c ac id which forms an ester-type g lucuronide , the h a l f - l i f e of which i s 2 to 6- fo ld longer i n renal f a i l u r e pat ients compared to healthy subjects (Gugler et a l . , 1979b). Another example of a drug which forms es ter- and ether-type glucuronides i s d i f l u n i s a l , a r ecent ly developed s a l i c y l a t e ana lges i c . - 158 -In a recent s ing le dose study, i t was shown that the t o t a l body clearance of d i f l u n i s a l was s i g n i f i c a n t l y decreased i n pat ients with r ena l f a i l u r e (De Schepper et a l . , 1977; Verbeeck et a l . , 1979). Poss ib le mechanisms include a l tered g lucuronidat ion of d i f l u n i s a l i n rena l f a i l u r e , b i l i a r y excret ion of one or both of the glucuronides followed by hydro ly s i s i n the i n t e s t i n a l t rac t and reabsorpt ion of unchanged d i f l u n i s a l , and systemic deconjugation of one or both of the glucuronides as has been described for the ester glucuronide of c l o f i b -rate (DeSchepper et a l . , 1977; Levy, 1979; Faed, 1980). Since toca inide also forms an ester-type g lucuronide , the increased h a l f - l i f e (2.5 fo ld) of the parent drug may be due to one or more of the above mechanisms operating i n the pat ient with renal dys funct ion . There has not been any report about s t e r e o s p e c i f i c i t y of drug d i s p o s i t i o n i n renal i n s u f f i c i e n c y . The present study also does not ind ica te the occurrence of s t e r e o s p e c i f i c i t y s ince the h a l f - l i v e s of both the isomers are increased to the same extent (Table 29). 5.3 Hemodialysis Hemodialysis i s the most common method of removing endogenous waste mater ia l i n chronic rena l f a i l u r e pa t i ent s . Factors such as blood f low, d i a l y s a t e flow and surface area of the d i a l y s i s membrane w i l l in f luence d i a l y s a b i l i t y . A decrease i n each of these factors w i l l tend to decrease the extent to which a drug i s d i a l y s e d . The pharmacokinetic c h a r a c t e r i s t i c s of a drug w i l l also have a s i g n i f i c a n t impact on the a b i l i t y of a drug to be d i a l y s e d . Those drugs that have a large volume of d i s t r i b u t i o n or are h igh ly prote in bound tend to be poorly d i a l y s e d . - 1 5 9 -The use of binding and d i s t r i b u t i o n data w i l l give one a general apprec ia t ion of the d i a l y s a b i l i t y of a given drug, but more prec i se estimates of the amount removed may be des i rab le for the purpose of dose adjustments. One approach involves the use of h a l f - l i v e s of drugs during d i a l y s i s , ( t j / 2 ) d , and when d i a l y s i s i s not being performed, ti/2» The f r a c t i o n of the drug removed by d i a l y s i s , f , i s given by f = h n - ^ i n h ( 1 _ e - ° - 6 9 3 t / ( t i / 2 ) d ) '1/2 where t i s the durat ion of d i a l y s i s (Gwi l t et a l . , 1978). The amount of a drug that w i l l be removed by d i a l y s i s , X^, can be ca lcu la ted i f the volume of d i s t r i b u t i o n , V , and p r e d i a l y s i s concentra t ion , C are known. X d = f . c v This amount can then be replaced after d i a l y s i s . Tocainide i s not prote in bound to any great extent (about 10%) ( E l v i n et a l . , 1982; Sedman et a l . , 1982) and i t had been shown that about (25 ± 14%) of the drug was removed by d i a l y s i s for 4 hours ® ( p a r a l l e l - p l a t e d i a l y s e r , ER -85, with c e l l u l o s e membrane, (surface area,, 1.2 m ) blood flow of 150-250 mL/min and a d i a ly sa te flow of 550 mL/min) (Wiegers et a l . , 1983). In the present study i n one pa t ient , the f r a c t i o n of tocainide enantiomers that would be removed by d i a l y s i s was 0.353, which was ca l cu l a ted by s u b s t i t u t i n g the fo l lowing h a l f - l i f e values i n the above equat ion: (S(+)T0C, t i / i = ^ 5 « 3 h r s » ^ 1 / 2 ^ = 6 , 5 n r s a n d 1 = 5 h r s ) . The amount of S(+) isomer removed was ca lcu la ted by the fo l lowing equat ion: - 160 -X D = 0.353 x 0.204 x 338 = 24.113 mg This corresponds to removal of 35% of the drug i n the body j u s t before d i a l y s i s . The p r e d i a l y s i s concentrat ion of S(+) isomer i n the a r t e r i a l plasma was 0.204 ug/mL, and the volume of d i s t r i b u t i o n of th i s isomer was 338 l i t e r s . S i m i l a r l y , the f r a c t i o n of the R(-) isomer removed by d i a l y s i s was 0.385 and the amount removed was 20.938 mg. 5.3.1 Clearance Calculations During Hemodialysis The log a r t e r i a l plasma concentration-time curve obtained during the 5 h hemodialysis i s shown i n Figure 33. The d i a l y s i s clearance was ca lcu la ted by the fo l lowing formula: c i ™ = Q A DP x p CA A where 0 1 ^ = d i a l y s i s plasma clearance = concentrat ion of drug i n a r t e r i a l plasma = concentrat ion of drug i n venous plasma Qp = plasma flow rate through the d i a l y s e r = Q b (1 - H) where = blood flow rate H = hematocrit . The fo l lowing table shows the a r t e r i a l and venous concentrat ions of both the isomers of tocainide and the A-V di f ference (Table 30) . - 1 6 1 FIGURE 33 LOG ARTERIAL PLASMA CONCENTRATION-TIME CURVE DURING 5HR HEMODIALYSIS a. < cc K-Z UJ o z o o < CO < _ l OL • S ( + ) t o c a i n i d e r=0.9626 fi =0.1055 h r t, =6.5 hrs h -1 A — A R ( - ) t o c a i n i d e -1 TIME (HRS) Table 30 A r t e r i a l and venous concentrations of the isomers of toca inide and the A-V d i f f e r e n c e . D i a l y s i s time (h) a r t e r i a l venous A-V d i f ference (+)T0C (pg/mL) (-)TOC (pg/mL) (+)T0C (pg/mL) (-)TOC (pg/mL) (+)T0C (-)TOC 1.0 0.1922 0.1764 0.1760 0.1379 0.0162 .0385 2.0 0.1893 0.1778 0.1640 0.1330 0.0253 0.0448 3.0 0.1700 0.1400 - - -4.0 0.1389 0.1249 - - - -5.0 0.1324 0.1112 0.1150 0.0671 .0174 .0441 - 163 -At a blood flow rate of 200 mL/min and a hematocrit value of 13.7, the plasma flow rate i s 172.6 mL/min. Subs t i tut ing the value of Qp and A-V di f ferences i n the above equation the fo l lowing d i a l y s i s c learances were obta ined. D i a l y s i s Clearance (mL/min) Time (hrs) (+)T0C (-)TOC 1.0 14.5 37.6 2.0 23.0 43.5 5.0 22.7 68.4 Mean ± SD 20.0 ± 4.8 49.8 ± 16.3 6. Stereoselective S a l i v a r y Excretion of Tocainide Enantiomers i n Man None of the published studies on toca inide has examined the p o s s i b i l i t y of secre t ion of R ,S- toca in ide or i t s enantiomers into s a l i v a . Secret ion of other ant iarrhythmic drugs in to s a l i v a has been studied with such agents as procainamide (Koup et a l . , 1975), qu in id ine ( Jaf fe et a l . , 1975), disopyramide ( A i t i o et a l . , 1982), l i d o c a i n e (Barchowsky et a l . , 1982) and mexi le t ine (Beckett and Chidomere, 1976). Wide i n t e r - and in t ra - sub jec t v a r i a t i o n i n sa l iva/plasma r a t i o s were observed and the o v e r a l l c o r r e l a t i o n between s a l i v a and plasma concentrat ion was poor. In the present study, the object ive was not only to measure - 164 -s a l i v a l e v e l s of R, S-tocainide but to measure the i n d i v i d u a l isomers a f ter intravenous adminis t ra t ion of the racemate and to compare the enantiomer p r o f i l e with that of plasma and u r i n e . It was also of i n t e r e s t to determine whether s a l i v a r y secre t ion of toca inide r e f l e c t s plasma concentra t ion . C a l i b r a t i o n curve data and p r e c i s i o n of assay of s a l i v a are shown i n Table 31. Tocainide enantiomer l e v e l s i n the s a l i v a fo l lowing an intravenous in fus ion of 200 mg of the racemic drug are shown i n Table 32 and the s a l i v a to plasma r a t i o s are given i n Table 33. The s a l i v a p r o f i l e one hour after the intravenous dose i s shown i n Figure 34. Throughout the period of measurement the s a l i v a r y concentrations of both the isomers were higher than the corresponding plasma concentra t ions , with the d i f ference being greater for the R(-) isomer (Table 32) . In one vo lunteer , whose s a l i v a r y pH ranged from 6.9 to 7 .1 , the concentrat ion of the R(-) isomer i n s a l i v a reached 6- fo ld higher l e v e l s than that of plasma concentra t ion , while the d i f ference was only 3- fo ld for the S(+) isomer ( table 33) . In another vo lunteer , who had c o n s i s t e n t l y high s a l i v a r y pH (7.4 to 7.5) the highest s a l i v a to plasma r a t i o of R(-) toca in ide was 3.5 and for S(+) t o c a i n i d e , 1.7. Comparison of data i n these volunteers appeared to suggest a pH dependent excre t ion of toca in ide into s a l i v a with the r a t i o of sa l iva/plasma increas ing with decreasing pH. Tocainide i s a weak base (pka = 7.7) and therefore i s s l i g h t l y ioni sed i n a c i d i c s a l i v a . The sa l iva/plasma r a t i o of such weak bases u sua l ly exceed uni ty as has been reported for drugs such as procainamide and l i d o c a i n e (Koup et a l . , 1975; Barchowsky et a l . , Table 31 C a l i b r a t i o n curve data and p r e c i s i o n of assay of s a l i v a quant i ty of each enantiomer (ng) area r a t io s ± S .C . S(+)T0C RSD(%) R(-•)T0C RSD(%) 50.0 0.1852 ± 0.0111 5.9 0.1704 ± 0.0115 6.7 100.0 0.2542 ± 0.0083 3.2 0.2334 ± 0.0106 4.5 200.0 0.4398 ± 0.0121 2.7 0.4435 ± 0.0130 2.9 500.0 1.0903 ± 0.0238 2.1 1.0963 ± 0.0249 2.2 750.0 1.5812 ± 0.772 4.8 1.6095 ± 0.816 5.0 1500.0 3.0566 ± 0.0785 2.5 3.1059 ± 0.0786 2.5 C o r r e l a t i o n c o e f f i c i e n t , r = 0.9998 0.9998 slope = 1.9987 .2.0446 intercept = --0.0685 0.0534 The quanti ty of i n t e r n a l standard was 1 ug. Table 32 Tocainide enantiomers in the saliva (ug/mL) of healthy subjects following an intravenous infusion of 200 mg (±) tocainide hydrochloride. Time (hrs) KM JA SR RE CK pH (+)T0C (-)TOC pH (+)T0C (-)TOC pH (+)T0C (-)TOC pH (+)T0C (-)TOC pH (+)TOC (-)TOC 0.75 7.1 0.766 1.404 7.4 0.935 1.837 - - - 7.1 1.360 2.337 - - -1.0 7.2 0.926 1.570 7.4 0.778 1.642 7.0 1.548 2.122 6.9 1.339 2.502 7.4 1.305 1.964 1.5 7.1 1.326 1.874 7.5 0.793 1.468 - . - 6.9 1.191 2.130 - - -2.0 7.2 1.167 1.475 7.5 0.672 1.212 7.1 0.986 1.638 6.9 1.031 1.724 7.3 0.734 1.077 3.0 7.4 1.019 1.379 7.5 0.431 0.618 6.9 0.843 1.245 6.9 1.431 2.075 7.1 0.734 1.077 5.0 7.1 1.150 1.432 7.3 0.548 0.824 7.0 0.815 1.209 6.9 1.186 1.489 - - -7.0 6.9 0.905 1.107. 7.0 0.529 0.802 6.9 0.905 1.417 6.9 0.562 0.595 7.6 0.775 1.033 10.0 - - - 7.2 0.113 0.148 7.0 0.926 1.418 7.0 0.400 0.475 7.1 0.665 0.885 24.0 7.1 0.372 0.408 7.4 0.208 0.314 7.0 0.289 0.380 7.1 0.333 0.288 7.2 0.173 0.222 30.0 7.3 0.269 0.259 7.5 0.147 0.252 7.0 0.138 0.179 6.9 0.210 0.201 7.3 0.165 0.158 36.0 7.5 0.166 0.085 - - - - - - - 7.6 0.123* 0.146* 48.0 7.3 0.094 0.065 - 7.2 0.068 0.056 7.0 0.091 0.032 7.5 0.064 0.063 *32.5 hrs. Table 33 Saliva/plasma concentration ratios of tocainide enantiomers i n healthy volunteers. Time KM JA SR RE CK (hrs) pH (+)T0Cs (+)T0Cp (-)TOCs (-)TOCp pH (+)TOCs (+)TOCp (-)TOCs (-)TOCp pH (+)TOCs (+)TOCp (-)TOCs (-)TOCp pH (+)TOCs (+)TOCp (-)TOCs (-)TOCp pH (+)TOCs (+)TOCp (-)TOCs (-)TOCp 0.75 7.1 0.766 1.404 7.4 0.935 1.837 - - 7.1 1.360 2.337 - - -1.00 - - - 7.4 1.473 3.547 6.9 3.086 6.257 7.0 3.005 4.505 7.4 2.926 4.453 1.50 7.1 2.702 4.413 7.5 1.653 3.415 6.9 3.117 6.136 - - - - - -2.00 7.2 2.828 4.339 7.5 1.305 2.616 6.9 2.769 5.199 7.1 2.176 4.015 7.3 2.723 4.724 3.00 7.4 2.280 3.560 7.5 1.053 1.623 6.9 3.788 6.447 6.9 1.821 2.971 7.1 1.835 2.834 5.00 7.1 2.721 4.386 7.3 1.251 2.119 6.9 3.362 5.350 7.0 2.124 3.754 - - -7.00 6.9 2.409 3.628 7.0 1.658 2.845 6.9 1.956 2.843 6.9 3.067 6.215 7.6 1.992 2.724 10.00 - - - 7.2 - - 7.0 1.492 2.605 7.0 3.379 6.534 7.1 1.837 2.431 24.00 7.1 1.771 3.043 7.4 1.400 3.060 7.1 2.088 4.142 7.0 1.783 3.551 7.2 0.789 1.982 30.00 7.3 1.509 2.387 7.5 1.137 3.516 6.9 1.742 4.827 7.0 1.422 3.140 7.3 1.122 2.135 36.00 7.5 1.179 1.183 - - - - - - - - - 7.3 1.122* 2.135* 48.00 7.3 1.002 1.505 - - - 7.0 3.095 7.2 1.658 4.000 7.5 0.771 1.575 TOCs = tocainide in saliva; TOCp - tocainide in plasma *32.5 hrs. - 168 -FIGURE' 34 . TOCAINIDE ENANTIOMERS saliva profile one hour after intravenous infusion of racemate 1. S(+) Tocainide 2. R(-) Tocainide 3. internal standard Blank saliva 0 10 20 M I N U T E S Chromatographic condi t ions : Column, C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y (50 m x 0.3 mm); i n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; c a r r i e r gas (H2) flow, 1 mL/min; make-up gas (N2) flow, 30 mL/min; s p l i t vent f low, 30 mL/min; column i n l e t pressure, 151.7 kPa; chart speed, 0.3 cm/min. - 169 -1982). The two fo ld d i f ference i n the r a t i o of the enantiomers as observed i n a l l the f ive volunteers cannot be explained on the basis of pH dependent i o n i s a t i o n or simple passive d i f f u s i o n . The high concentrat ion of the R(-) isomer i n the s a l i v a as compared to S(+) isomer i s the opposite of the plasma or ur ine p r o f i l e at the same time p e r i o d . However, the enantiomer r a t i o i n the s a l i v a tends to approach that of plasma and ur ine p r o f i l e by 48 hours (Figure 35) (Table 34) . This r ever sa l of s a l i v a p r o f i l e [R(-) > S(+) to (S(+) > R(-)] a l so cannot be explained on the basis of the known mechanisms of drug excre t ion into s a l i v a . However, the data i s consis tant with the observat ion that the R(-) isomer disappears fas ter from the plasma and s a l i v a as shown by i t s shorter h a l f - l i f e . It appears that the R(-) isomer i s r a p i d l y d i s t r i b u t e d i n i t i a l l y in to the s a l i v a at twice the rate of S(+) isomer and thereaf ter the concentrat ion of the R(-) isomer i n s a l i v a f a l l s at a much greater rate than the S(+) isomer. As a r e s u l t the concentrat ion of the R(-) isomer i s less than the concentra-t i o n of S(+) isomer i n most volunteers a f ter 24 hours . The sa l iva/plasma concentrat ion of a basic drug can be predic ted according to the equation: 1 + 10 ( p k a - P H S ) s/p = ( p k a - P H p ) x f / f p s 1 + 10 where pk a i s the p k a of the bas ic drug that i s transported from plasma to s a l i v a , pH p i s the pH of the plasma, pH g i s the pH of the - 170 -FIGURE 35 L O G P L A S M A A N D S A L I V A C O N C E N T R A T I O N S v s T I M E F O R R ( - ) - A N D S (+ ) - T O C A I N I D E E N A N T I O M E R S 3 CO z o t-< cc t-z LU o z o o < > < CO o z < < CO < o o S(+)tocainide in saliva S(+)tocainide in plasma R(-)tocainide in saliva R(-)tocainide in plasma 20 30 40 T I M E ( H O U R S ) 50 Table 34 Enantiomer r a t i o , (+)T0C/(-)T0C, in plasma, urine and s a l i v a following intravenous infusion of 200 mg (±) tocainide hydrochloride to healthy male volunteers. Time (hrs) KM JA SR RE CK P* U* S* P 0 S P U S P U S P U S 0.33 1.04 - - 1.13 - - 1.09 - - 1.03 - - 1.00 - -0.75 1.12 - 0.54 1.03 - 0.51 1.07 - - 1.07 - 0.58 0.98 - -1.00 - 0.98 0.59 1.14 1.03 0.47 1.09 0.97 0.73 1.08 0.99 0.53 1.00 0.96 0.68 1.50 1.16 - 0.71 1.12 - 0.54 1.09 - - 1 .10 - 0.56 0.95 - -2.0 1.21 1.07 0.79 1.11 0.98 0.55 1.11 1.00 0.60 1.12 1.15 0.59 0.99 0.96 0.57 3.0 1.15 1.09 0.74 1.07 0.99 - 1.11 1.08 0.68 1.17 1.13 0.69 1.01 1.03 0.68 5.0 1.29 1.05 ' 0.80 1.12 1.06 0.66 1.14 1.09 0.68 1.26 1.14 0.79 1.04 1.05 0.80 7.0 1.23 1.08 0.82 1.13 1.16 0.66 1.29 1.16 0.64 1.37 1.20 0.94 1.03 1.11 0.75 10.0 - 1.10 0.77 1.23 1.17 - 1.26 1.17 0.65 1.47 1.33 0.84 1.14 1.06 0.75 24.0 1.59 1.31 0.92 1.45 1.31 0.66 1.51 1.30 0.76 2.29 1.88 1.15 1.95 1.35 0.78 30.0 1.64' 1.40 1.04 1.80 1.45 0.58 1.70 1.38 0.77 2.90 2.30 1.04 1.99 1.61 1.04 48.0 2.16 1.52 1.45 2.45 2.00 - 2.89 1.84 1 .21 - 3.50 2.82 2.07 2.00 1.02 72.0 - 2.07 - - 2.26 - - 2.73 - - - - - 2.28 -*P = plasma *U = urine *S = Saliva - 172 -s a l i v a at the time of secre t ion of the drug, fp i s the f r a c t i o n of the drug unbound i n plasma and f s i s the f r a c t i o n unbound i n s a l i v a which i s u sua l ly taken as u n i t y . Tocainide i s not bound to plasma proteins to any great extent . E l v i n et a l . , (1982) have shown that the unbound f r a c t i o n of toca in ide i n the serum of 10 healthy volunteers ranged from 0.78 to 0.96 and from 0.8 to 0.9 i n 4 trauma pat ients (who had high concentrat ions of a ^ a c i d g l y c o p r o t e i n ) . Binding data consis tent with the above study were reported by Sedman et a l . (1982) using serum samples from 5 heal thy vo lunteer s . The percentage of t o t a l unbound toca inide ranged from 85 to 90% and binding appeared to be independent of serum concentrat ion wi th in the range of 4-12 ug/mL. Using f p = 0 .9 , pk a = 7.7 and pHp 7.4 i n the above equat ion, the sa l iva/plasma r a t i o of toca in ide has been predicted and compared with the r a t i o observed ( table 35). The observed r a t i o s were always higher than predicted and the c o r r e l a t i o n was poor. Although there was a marked inter sub jec t v a r i a b i l i t y between the sa l iva/plasma r a t i o s (1.406 to 2.603 with a mean of 2.068 ± 0.508 (SEM) for S(+) toca in ide ; 2.914 to 4.374 with a mean of 3.683 ± 0.760 (SEM) for the R(-) isomer, the c o r r e l a t i o n between the s a l i v a and plasma concentrat ion i n the same i n d i v i d u a l was good, (r = 0.910 to 0.987 for S(+) isomer; 0.884 to 0.986 for the R(-) isomer) ( F i g . 36). 6.1 Tocainide Enantiomers i n the S a l i v a of a Patient with Renal  Dysfunction The s a l i v a r y concentrations of S(+) and R(-) toca in ide were measured i n the s a l i v a of a patient with t o t a l renal f a i l u r e (who was - 173 -Table 35 Comparison of observed and predicted sa l iva/plasma r a t i o s of S(+) tocainide i n one of the healthy volunteers (KM) Predicted s/p r a t i o Time (hrs) pH Observed s/p r a t i o — Uncorrected Corrected 0.75 7.1 1.458 1.662 1.496 1.50 7.1 2.702 1.662 1.496 2.0 7.2 2.828 1.055 0.950 3.0 .7.4 2.280 1.000 0.900 5.0 7.1 2.721 1.662 1.496 7.0 6.9 2.409 2.440 2.196 24.0 7.1 1.771 1.662 1.496 30.0 7.3 1.509 1.172 1.055 36.0 7.5 1.179 0.862 0.776 48.0 7.3 1.002 1.172 1.055 - 174' -FIGURE 36 CORRELATION BETWEEN SALIVA AND PLASMA LEVELS OF TOCAINIDE ENANTIOMERS FOLLOWING INTRAVENOUS INFUSION OF R.S-TOCAINIDE !.0 E cn U z o o < > < CO 1.5 1.0 0.5 S(+) Tocainide r=0.9289 0.1 "02" "03" 0.4 0.5 PLASMA CONC. ug/mL - 175 -undergoing hemodialysis three times a week) fo l lowing intravenous adminis t ra t ion of the racemic drug. Plasma l e v e l s were also measured at the same time periods as for s a l i v a . The sa l iva/plasma r a t i o s of both the isomers are given i n Table 36. The s a l i v a r y concentrations of both the isomers as wel l as the sa l iva/plasma ra t io s were very high i n th i s pat ient compared to healthy vo lunteer s . It appears from th i s data that the sa l iva/plasma r a t i o of th i s weakly basic drug (pk a = 7.7) i s not governed by pH of the s a l i v a (pH was cons i s tent ly h igh , 8.0 - 8.4 throughout the period of measurement (24 h r s ) . D. Analysis of Tocainide Metabolites i n the Urine Tocainide has been reported to be metabolized i n man by a novel pathway to a carbaminic acid followed by conjugation with g lucuronic ac id ( E l v i n et a l . , 1980b). This g lucuronide , unl ike any prev ious ly reported i s thought to undergo c y c l i s a t i o n at pH 13 to a hydantoin-type compound ( E l v i n et a l . , 1980b). TOCG belongs to the group of ester-type glucuronides where a carboxy group i s conjugated with g lucuronic a c i d . Conjugates of var ious n o n - s t e r o i d a l antiinflammatory drugs such as indomethacin, s a l i c y l i c ac id and ketoprofen are examples of e s te r -g lucuronides . The ester - 176 -Table 36 pat ient Tocainide enantiomers : with renal dysfunct ion i n the s a l i v a and s a l i v a / p l of a asma r a t i o s • Time (hrs) (+)T0C (Ug/mL) (-)TOC (ug/mL) r a t i o (+)T0C g ( - )T0C 8 pH (+)T0C P (-)TOC P • 1.0 8.4 5.202 6.030 0.862 - -2.0 8.3 5.110 6.067 0.842 - 19.320 3.0 8.3 4.187 4.371 0.957 12.103 13.407 5.0 8.4 3.655 4.303 0.849 10.563 18.708 7.0 8.0 1.763 1.923 0.916 - -10.0 8.1 1.204 1.123 1.072 4.613 4.925 12.0 8.1 0.957 0.940 1.018 3.922 4.747 24.0 8.0 0.419 0.386 1.085 2.073 2.590 T0C s = toca in ide i n s a l i v a TOCL, = toca inide i n plasma - 177 -glucuronides i n general are unstable i n a l k a l i n e so lu t ions and also during storage i n the freezer (Upton et a l . , 1980). For example, ketoprofen glucuronide i s known to hydrolyse slowly when the urine i s stored at - 1 5 ° C (Upton et a l . , 1980a). Another problem with ester glucuronides i s intramolecular acy l migra t ion . The aglycone can migrate from p o s i t i o n 1 to 2,3 or 4. Recent reports suggest such migrat ion for zomepirac, probenecid and ketoprofen (Hasegawa et a l . , 1982; Upton et a l . , 1980a; Eggers and Doust, 1980). Only one of the four poss ib le glucuronides i s hydrolysed by the enzyme,*,, 3-glucuronidase. The fact that a l l glucuronides are not hydrolysed by the commonly used 3-glucuronidase must be considered while developing assay methods for g lucuronides . Tocainide carbamoyl glucuronide i s excreted i n the human ur ine to the extent of about 30% of the administered dose w i t h i n the f i r s t 72 hours ( E l v i n et a l . , 1980a). Since toca inide has one c h i r a l center , conjugation with g lucuronic ac id w i l l r e su l t i n the formation of two diastereomeric g lucuronides . It i s genera l ly recognized that g lucuronida t ion i s a s tereose lect iveprocess for c h i r a l compounds. Since a subs tan t i a l f r a c t i o n of toca in ide dose i s excreted as g lucuronides , i t i s of considerable in te re s t to e s t a b l i s h whether s t e reose lec t ive g lucuron ida t ion occurs with th i s drug and also whether disease states such as rena l dysfunct ion a l t e r t h i s process . Such a study necess i ta tes an a n a l y t i c a l technique to measure the enantiomeric glucuronides,. 1. Gas Chromatographic Analysis of Glucuronides Glucuronides have been character ized by examination of the aglycone released by ac id or enzymatic h y d r o l y s i s . Widespread concern - 178 -e x i s t s about the s e l e c t i v i t y of both enzymic or chemical hydro ly s i s and about the wide v a r i a t i o n of t h e i r rates of hydro lys i s and the optimal condi t ions with d i f f e r e n t substrates . The s tructure of the aglycone may be a l tered by chemical hydro lys i s and the enzyme, 3 _ glucuronidase , may not hydrolyse isomeric glucuronides to the same extent (Hasegawa et a l . , 1982). Thus hydro ly s i s i s not r e l i a b l e e i ther q u a n t i t a t i v e l y or q u a l i t a t i v e l y , and the r a t i o n a l e c l e a r l y ex i s t s for ana lys i s of i n t a c t g lucuronides . In recent years chromatographic techniques have been appl ied to the c h a r a c t e r i s a t i o n of i n t a c t g lucuronides . Glucuronides are polar po ly funct iona l molecules that usua l ly undergo thermal degradation i n GC systems p r i o r to v o l a t i l i z a t i o n . It i s therefore necessary to prepare v o l a t i l e , thermally stable d e r i v a t i v e s . Frequent procedures are methyla t ion , a c e t y l a t i o n , t r i f l u o r o a c e t y l a t i o n and t r i m e t h y l s i l y l a t i o n . In a ser ies of papers, Thompson et a l . , (1972, 1973, 1975) described a one-step procedure for the methylat ion of glucuronides and t h e i r ana lys i s by gas chromatography/mass spectrometry. Many c h a r a c t e r i s t i c ion fragments der ived from the permethylated g lucuronic acid port ion such as m/z 232, 201, 169, 141, 101 and 75 can be observed i n the mass spectra . However, g lucuronides of the ester-type are known to be p a r t i a l l y hydrolysed during the process of permethylation (Thompson et a l . , 1973). Decarbamylation takes place i n the case of carbamazepine glucuronide (Lynn et a l . , 1978). 2. Liquid Chromatographic Analysis of Glucuronides L i q u i d chromatography of fers an a t t r a c t i v e a l t e r n a t i v e to gas chromatography for the i s o l a t i o n and assay of n o n - v o l a t i l e and polar - 179 -metaboli tes such as g lucuronides . No d e r i v a t i s a t i o n i s required and the assay i s conducted at room temperature. Although the g lucuronic acid moiety does not contr ibute to u l t r a v i o l e t absorpt ion , many aglycones do have some chromophoric groups. Separation of Diastereomeric Glucuronides The formation of a glucuronide from a c h i r a l molecule i s s imi l a r to d e r i v a t i s a t i o n i n GC with o p t i c a l l y act ive reagents to form diastereomers . Since diastereomers have d i f ferences i n the ir phys ica l p roper t i e s , i t i s poss ible to resolve them by l i q u i d chromatography. Using a reverse-phase column (Cj^) and 8% a c e t o n i t r i l e i n phosphate buffer (pH 7.5) two diastereomeric glucuronides of hydantoin have been resolved (Hermansson et a l . , 1982). A s i m i l a r r e s o l u t i o n also has been reported for propranolol glucuronides employing an RP-18 column and methanol/ammonium phosphate as the mobile phase (Thompson et a l . , 1981). 3. I s o l a t i o n of Glucuronides from Prine Solvent ex t rac t ion methods are not very su i t ab le for the i s o l a t i o n of g lucuronic acid conjugates from b i o l o g i c a l m a t e r i a l s . Act iva ted charcoal i s sometimes used to adsorb g lucuronic acid conjugates. A more commonly used adsorbent for polar metabolites from aqueous so lu t ions i s XAD-2 r e s i n . Amberlite XAD-2 r e s i n i s nonionic , s t y r e n e - d i v i n y l benzene co-polymer having a large surface area. I t s most s i g n i f i c a n t property i s the adsorption of water soluble organic species from aqueous s o l u t i o n s . XAD adsorption does not separate the parent drug from i t s - 180 -metabolites but i s a useful concentrat ion step. From a large volume of ur ine the drug and metabolites can be obtained i n a concentrated form i n organic so lvents . I t i s app l icab le to a l l types of glucuronides and i s u n l i k e l y to cause any undesirable s t r u c t u r a l a l t e r a t i o n of the conjugates. Fujimoto and Wang (1970) were two of the f i r s t inves t iga tor s to descr ibe a simple procedure for the ex t rac t ion of narcot ic analgesics from urine with XAD-2 r e s i n . Thei r ear ly work showed that ur inary pigments were also adsorbed on to the r e s i n and were eluted i n the same f r a c t i o n as the drugs and metabol i tes . Thus, the f r a c t i o n with the greatest colour i n t e n s i t y a l so corresponded to the f r a c t i o n conta ining the greatest amount of drugs and the ir metabol i tes . Bradlaw (1968) reported a successful and simple i s o l a t i o n procedure using neutra l XAD-2 r e s i n for the ex t rac t ion of s t e ro id glucuronides from u r i n e . The i s o l a t i o n and c h a r a c t e r i z a t i o n procedure for tocainide glucuronide involved four major steps (Figure 37) : 1. Adsorption on XAD-2 r e s i n . 2. Thin l ayer chromatography on a preparat ive scale us ing reverse-phase plates and i s o l a t i o n of bands corresponding to g lucuronide . 3 . Permethylat ion and analys i s by gas chromatography/mass spectrometry. 4 . D i r e c t ana lys i s by l i q u i d chroraatography/mass spectrometry. - 18-1 -FIGURE 37 I S O L A T I O N OF T O C A I N I D E GLUCURONIDES FROM HUMAN U R I N E u r i n e I e l u t e w i t h H y e l u t e w i t h Me 0 OH MEOH EXT.' vac. evaporate, freeze dry F R E E Z E D R I E D SAMPLE I N NaOH HYDANTOIN , MEOH PH=13 D E R I V A T I V E I GC, FID > LC GC/MS T L C V I S U A L I Z E BANDS MEOH: H 2 0 WITH NAPHTHORESORCINOL 6 0 : 4 0 SPRAY REAGENT GC, FID LC GC/MS HYDANTOIN D E R I V A T I V E NaOH pH=l: I I S O L A T E BANDS CORRESPONDING TO GLUCURONIDES 6 - g l u c o u r o n i d a s e pH=5. GC, ECD<-T O C A I N I D E ISOMERS V Hcl LC/MS H 2 0 : C H 3 C N 9 0 : 1 0 100° P E R M E T H Y L A T I O N DMSO, C H 3 I r GC/MS - 182 -A. Micropore LCMS of Tocainide Glucuronide The p o s i t i v e ion mass spectrum obtained i n the chemical ionisa-tion mode i s shown in Figure 38. The ion at m/z 236 corresponds to a ft ft HCCHNHCOH m/z 236 structure which i s the aglycone of tocainide carbamoyl glucuronide. The ion at ra/z 219 was formed either by loss of an -OH group leading to the acylium Ion, or by c y c l i s a t i o n to a hydantoin structure as shown below: M/Z 2 1 9 K/Z 2 1 9 A c y l i u m i o n H y d a n t o i n Formation of 'acylium' ions from esters (Budzikiewicz et a l . , 1967) and ester-glucuronides (Baake et a l . , 1976) have been reported. The po s i t i v e ion spectra also contained fragments such as m/z 197, m/z 181, m/z 165, m/z 162 (base peak) and m/z 153 but a structure for these ions - 183 -FIGURE <38; ' MICROBORE LCMS OF TOCAINIDE GLUCURONIDES i6a 528^ 236 H Q H H O | N -C -C -N -C—^ 1 I O II C-OH CH, CH, IX OH OH 36&\ N carboxy tocaimda glucuronic)* MW • 412 280-1 219 236 208^ 120H 153 4B-X 165 181 197 / 200 160 180 220 - 1 8 4 -could not be r a t i o n a l i z e d . The negative ion spectra contained only one fragment, m/z 119. The same ion (m/z 119) was also the base peak i n the negative ion spectra of p-ni trophenol glucuronide obtained under i d e n t i c a l c o n d i t i o n s . In addi t ion to m/z 119, this spectra a lso contained m/z 139 which corresponds to p-n i t rophenol . Cairns and Siegmund (1982) have shown that the base peak obtained for g lucuronic ac id at a source temperature of 2 2 0 ° C was the dehydrated product, m/z 119 and that the pattern of fragmentation was temperature s e n s i t i v e . Since the spectra of p-nitrophenol glucuronide (Figure 39) and tocainide glucuronide (Figure 38) contained m/z 162 ion (+ve ion mode), as we l l as m/z 119 (-ve ion mode), i t i s bel ieved that both of these ions were derived from the g lucuronic acid moiety. Thus, a microbore LCMS study added more d i r e c t evidence for the proposed s tructure of tocainide carbamoyl g lucuronide . 5. I d e n t i f i c a t i o n of Band l b as Tocainide Carbamoyl-O- B-D-Glucuronide The f r a c t i o n l a b e l l e d as band 1^ was converted to a hydantoin-type compound by adjust ing the pH of the so lu t ion to 13 ( E l v i n et a l . , 1980). F igure 40 shows the chromatogram of the hydantoin derived from a glucuronide of tocainide along with standard hydantoin d e r i v a t i v e supplied by Astra Pharmaceuticals . The i d e n t i t y of the hydantoin was further proved by comparison of the r e t en t ion times and fragmentation patterns obtained by c a p i l l a r y column gas chromatography/ mass spectrometry of the reference substance and the i so l a t ed mater ia l (F igure 41 and 42) . - 185;..^  FIGURE 3 9 MICROBORE LCMS OF p-NITROPHENOL GLUCURONIDE 157 lseia-1000-COOH 5BB 162 MW 315 ..L 181 198 19S 155 165 175 185 - 186 -FIGURE 40 HPLC OF HYDANTOIN (STD)(A) AND HYDANTOIN DERIVED FROM A GLUCURONIDE OF TOCAINIDE(B) Column:ODS,mobHe phase • 25% Acetonitrile in 0.05 m. pot.chlorate. - 1 8 7 -r FIGURE 41 GC OF HYDANTOIN(STDXA) AND HYDANTOIN DERIVED FROM A GLUCURONIDE OF TOCAINIDE (B) e column:SE-30 fused silica capillary(l5m»0.25mm) isothermal at 190 Chromatographic cond i t ions : In j ec t ion temperature, 2 4 0 ° C ; detector (FID) temperature, 3 0 0 ° C ; oven temperature, 1 9 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; make-up gas (He) flow, 60 mL/min; s p l i t vent flow, 30 mL/min; column i n l e t pressure, 55.1 kPa; chart speed, 0.3 cm/min. - 1 8 8 - . FIGURE .42 " CI MASS SPECTRA OF A HYDANTOIN DERIVATIVE OBTAINED FROM A GLUCURONIDE OF TOCAINIDE 219 108 (M+1) S c a n 8 . 8 5 m in . 148 CH. 0 CH, O CH> MW 218 203 i a a i z a i4-a i6a tea 2ee 220 z*a zee ZBB - 189 -The f r a c t i o n l a b e l l e d as band I b also gave tocainide after h y d r o l y s i s with ft-glucuronidase or 1 N HC1 ( F i g . 43) . This chromatogram showed that more of the R(-) isomer i s generated by hydro ly s i s and that there i s more of R(-) tocainide glucuronide i n urine than S(+) toca inide g lucuronide . This was further supported by a more s e l ec t ive hydro lys i s of the urine as we l l as band 1^ with ^-glucuronidase enzyme. After h y d r o l y s i s , the quanti ty of R(-) tocainide was increased compared to the p r o f i l e of unhydrolysed u r i n e . Band I a d id not give tocainide after acid or enzyme h y d r o l y s i s . 6. Gas Chromatographic/Mass Spectrometrlc Analysis of Permethylated  Glucuronides Frac t ions i s o l a t e d by preparat ive TLC as wel l as the freeze dr ied methanolic extract of urine were, permethylated for gas chromatographic a n a l y s i s . 6.1 CI and EI Mass Spectra of Permethylated Glucuronic Acid The CI mass spectrum of permethylated g lucuronic acid i s presented here as a reference to aid i n the i n t e r p r e t a t i o n of permethylated glucuronide mass spectra (Figure 44, 45 and Table 37) . There was no molecular ion present at m/z 264. The highest mass ion was at m/z 293 which corresponds to M + C2H.5 + (probable when methane i s used as reactant gas). The i o n , m/z 233 corresponds to los s of a methoxy group (M -OCH3). The fragment ion m/z 233 then loses MeOH success ive ly - 190 -FIGURE -43 CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVES OF TOCAINIDE ENANTIOMERS OBTAINED BY ACID-HYDROLYSIS OF BAND Iv, 1. S (+) toca in ide 2. R (-) toca in ide Chromatographic cond i t ions : Column, C h i r a s i l - V a l ® f u s e d - s i l i c a c a p i l l a r y (50 m x 0.3 mm); i n j e c t i o n temperature, 2 4 0 ° C ; detector (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f low, 1 mL/min; s p l i t vent flow, 30 mL/min; make-up gas (N2) f low, 30 mL/min; chart speed, 0.3 cm/min. - 191 -FIGURE 44 GC/MS/CI OF PERMETHYLATED GLUCURONIC ACID COLUMN: SE-30 FUSED-SILICA CAPILLARY 2 0 6 0 8 8 «e 1 20 160 200 240 280 328 360 - 1 9 2 -FIGURE 45 F R A G M E N T A T I O N P A T T E R N O F P E R M E T H Y L A T E D G L U C U R O N I C A C I D C H 3 0 = C H O C H 3 M/Z 75 C H 3 0 = C H C H = C H O C H 3 M/Z 101 / C O O C H , o-CH36 | O C H 3 O C H , C O O C H O C H 3 / " y/-0CH3 MW 2 6 4 y -COOCHa OCH3 M/Z 2 3 3 - CH30H M/Z 201 CH30H M/Z 1 6 9 CH ,0 0 C H 3 OCH3 M/Z 2 0 5 CH 3 OH M/Z 173 C H 3 O H M/Z 141 - 193 -to give m/z 201 and m/z 169. The ions , m/z 101 and m/z 75 were formed by cleavage of the pyranoside r i n g . The m/z 201 ion was the most intense ion i n the CI mass spectrum (Table 37). On the other hand, m/z 101 was the most intense ion i n the EI mass spectrum. I n t e n s i t i e s of ions such as m/z 141, 88 and 75 were also increased i n the EI mass spectrum. The fact that ions such as m/z 205, 173 and 141 were formed only under e lec t ron impact i o n i s a t i o n suggested that removal of -COOCH3 group may be a prominent step involved under these condi t ions . 6.2 CI Mass Spectra of Permethylated p-nitrophenol glucuronide As shown i n the CI mass spectrum (Figure 46 and Table 3 8) the M + 1 ion at m/z 372 was the most intense i n the spectra . The highest mass ion at m/z 400 corresponded to M + C2H5+ and m/z 340 corresponded to M-OCH3. Fragments c h a r a c t e r i s t i c of the permethylated g lucuronic acid por t ion of the molecule such as m/z 233, m/z 201, m/z 169, m/z 145, were a lso seen i n the spectrum. The i o n , m/z 139 was that of the aglycone, p-ni t rophenol which was not evident i n the EI mass spectrum. A d d i t i o n a l fragments such as m/z 173, 141, 88 and 75 were also formed under e l e c t r o n impact condit ions from the g lucuronic acid moiety. 6.3 GCMS of Permethylated Tocainide Glucuronides Using an SE-30  F u s e d - s i l i c a C a p i l l a r y Column Toca in ide glucuronides (band 1^ ) i s o l a t e d by preparat ive t h i n - l a y e r chromatography were permethylated and analysed by gas chromatography/mass spectrometry using an SE-30 f u s e d - s i l i c a c a p i l l a r y - 194 -Table 37 Re la t ive i n t e n s i t i e s of fragment ions of permethylated g lucuronic acid i n the CI and EI mass spec t ra . r e l a t i v e i n t e n s i t y % r e l a t i v e i n t e n s i t y % m/z CI EI 293 9 -263 3 -233 19 -205 - 2 201 100 1 173 - 2 169 50 5 145 3 4 141 - 2 101 11 100 88 - 29 75 8 36 - 195 -FIGURE 46 . Q C / M S / C I O F P E R M E T H Y L A T E D p - M I T R O P H E N O L O L U C U R O N I D E column : S E-30 fused s i l i c a c a p i l l a r y . s p l l t l e s s i n j e c t i o n , m e t h a n e r e a g e n t gas i s e see «ee - 196 -Table .38 Re la t ive i n t e n s i t i e s of fragment ions of permethylated p-ni trophenol glucuronide i n the CI and EI mass spec t ra . m/z r e l a t i v e i n t e n s i t y % CI r e l a t i v e i n t e n s i t y % EI 400 16 -372 100 -340 3 -308 9 -240 24 -232' 3 9 201 29 100 177 24 -173 - 7 169 4 14 145 10 3 141 - 27 139 4 -101 - 37 88 - 13 75 - 30 - 197 -column (25 m x 0.2 mm). The permethylated sample was in jec ted onto the column by the s p l i t l e s s mode and the oven was programmed from 5 0 ° to 1 4 0 ° at 3 0 ° / m i n and from 1 4 0 ° to 2 4 0 ° at 1 0 ° / m i n . The mass spectrometer was operated i n the EI mode. F igure 47A shows the t o t a l ion current p r o f i l e and F i g . 47B i s the E l mass spectra of the peak e l u t i n g at 18.9 minutes. The mass spectra of the three compounds e l u t i n g at 18.6, 18.9 and 19.4 mins. contained many of the same fragment ions but with varying i n t e n s i t y (Table 39) . A l l the three had most of the c h r a c t e r i s t i c fragments of a permethylated glucuronide (m/z 201, 141, 101, 88 and 75) but none showed a molecular ion which could be corre la ted with the s tructure of the glucuronide metabolites reported for tocainide (molecular ions of permethylated glucuronides of l a b i l e compounds are not always seen i n the EI spec t ra ) . Table 39 shows the i n t e n s i t i t e s of var ious Ion fragments for the three compounds. The ion at (m/z 277)which was found i n considerable abundance i n a l l the three spect ra , was probably derived from the permethylated ester glucuronide s ince such fragmentation i s known to occur for esters - 1 9 8 -FIGURE 47 G C / M S O F P E R M E T H Y L A T E D G L U C U R O N I D E S O F T O C A I N I D E column SE 30 fused •ll lca(is-m>ozs) spl i t less Injection s , m^2G0' oven temp. 50 5 mm 4 e ia 16 2B 24 28 32 36 43 44 100 80 6B 40 20 105 98 72 +4. EI Mode 18 93 min. 156 141 12J 2B1 ITl 10s C H , . -H 0 H H I II I I " - C - N I C H , _277 2 3 a * 201,141,101,75 0 II C-OCH, yO/rN~c-(f-N-(--Ov 0 1 \ C M > 0 /\ J *0CH, OCH, 277 232 _ i l _ 3aa i 334 iaa 20B 4 3 0 sea - 199 -Table 39 Re la t ive i n t e n s i t i e s of fragment ions of permethylated glucuronides of toca inide extracted from u r i n e . ( E l e c t r o n impact mode) Retention time: 18:6 mins 306(8) 277(10) 263(15) 248(8) 232(3) 215(39) 201(16) 187(63) 182(16) 172(10) 157(41) 156(37) 148(2) 141(23) 128(46) 121(7) 113(16) 105(73) 101(53) 98(100) 90(93) Retention Time, 18:9 mins 333(4) 302(3) 277(38) 236(3) 232(2) 203(2) 201(45) 172(17) 157(11) 156(77) 141(42) 131(15) 128(24) 116(17) 105(100) 101(18) 98(98) 88(31) 77(18) 75(20) 72(41) 46(23) 44(13) Retention Time 19:4 mins 467(2) 320(6) 277(9) 261(4) 235(5) 201(26) 172(9) 157(7) 156(48) 141(35) 128(12) 116(34) 101(26) 98(100) 90(78) 88(36) 72(32) 70(21) 46(16) 44(14) - 200 -(Budzikiewicz et a l . , 1967). The ion at ra/z 192 was not seen i n the spectrum (ra.w. of tocainide = 192). However, there was evidence for the presence of the ion: which was the most intense (100%) for the peak e l u t i n g at 18.9 mins. The three ion fragments, m/z 156, 157 and 98 were present i n a l l the spectra but a structure could not be d e f i n i t e l y assigned. The t o t a l ion current p r o f i l e ( F i g . 51a) also indicated that extensive degradation had taken place during d e r i v a t i s a t i o n of the ester glucuronide or during chromatographic a n a l y s i s . The compound e l u t i n g at 4.8 mins showed the following ion fragments: m/z 201, 141, 101, 88 and 75, a l l of which are c h a r a c t e r i s t i c of permethylated glucuronic a c i d , suggesting at l e a s t p a r t i a l deconjugation of the glucuronide. The mass spectrum of the peak e l u t i n g at 6.7 mins showed fragments such as m/z 176, 148, 105 and 77. The proposed structure of these ions are: m/z 105 HCCH m/z 176 m/z 148 m/z 105 m/z 77 - 2 0 1 -Therefore, this peak must be tocainide or derived from tocainide. The most intense Ion (100%) i n the mass spectra of compounds e l u t i n g at 8.4, 9.0, 9.2, 9.5, 10.1, and 10.2 minutes was m/z 135 which corresponds to: m/z 135 This fragment ion could have been formed by the raethylation of the amide nitrogen by"raethyliodide i n the presence of dimsylsodiura. However, there was no other evidence to suggest that such an a r t i f a c t arose during the d e r i v a t i s a t i o n process. 6.4 GCMS of Permethylated Tocainide Glucuronides Using a  C h i r a s i l - V a l * F u s e d - s i l i c a C a p i l l a r y Column Due to the close s i m i l a r i t y of the mass spectra of the three compounds e l u t i n g from the SE-30 c a p i l l a r y column a further i n v e s t i g a t i o n of the GC/MS c h a r a c t e r i s t i c s of th i s sample was made using the Chirasil-Val® f u s e d - s i l i c a column. Figure 48A shows the t o t a l ion current p r o f i l e of the permethylated f r a c t i o n and the EI mass spectrum of the peak e l u t i n g at 7.91 minutes i s shown i n Figure 48B. This peak yeilded abundant mass spectral evidence for the presence of glucuronide conjugate of tocainide structure assignments for those fragments at m/z - 202 -FIGURE 48 QC/MS/EI OF PERMETHYLATED GLUCURONIDE OF TOCAINIDE Column: Ch i ras i l -Va l ( 1 5mx0.31 mm) oo ^ o o "Z. 6. 6 7 -18 12 14- 16 B 2 en 121 176 192 277 CH3 \ \ Y NHCoicHNHiCOO : !| : 1 CH, COOCH, CH3 ,05 1 4 8 iCH 3 ^232 MW 468 101 7 S 45 29 56 »5 U 9* X 141 7 .91 min. 116 169 156 145 192 232 233 277 34B 40 TO 13° i6«> 200 240 asa 32e - 203 -192, 176, 148, 121, 105, 90 and 77 could be corre la ted with the tocainide por t ion of the molecule, while fragments at m/z 233, 201, 169, 101, and 75 could be corre la ted with those shown previous ly to ar i se from a permethylated sample of g lucuronic a c i d . Those peaks e l u t i n g at 9 .21, 9.80 and 11.49 minutes a l so exhibi ted a number of ions which were c h a r a c t e r i s t i c of a permethylated g lucuronide . However, evidence for the tocainide moiety was weak and hence probable s tructures were not ass igned. - 204 -SUMMARY AND CONCLUSIONS A new g a s - l i q u i d chromatographic assay method for measurement of low l e v e l s of ( ± ) tocainide i n b i o l o g i c a l f l u i d s employing a fused-s i l i c a c a p i l l a r y column and e lec t ron capture detect ion has been developed. The a p p l i c a b i l i t y of this technique has been demonstrated for pharmacokinetic studies i n small laboratory animals and also i n humans. The method has a l l the advantages associated with the use of i n e r t and f l e x i b l e f u s e d - s i l i c a c a p i l l a r y columns and the high r e s o l u t i o n c a p a b i l i t y inherent with the system, combined with s e l e c t i v e and s e n s i t i v e detect ion by e lec t ron capture d e t e c t i o n . The s te reose lec t ive assay method developed i s based upon the d i r e c t r e s o l u t i o n of r e a d i l y prepared hepta f luorobutyryl de r iva t ive s of toca inide enantiomers and e l ec t ron capture d e t e c t i o n . Determination of enantiomeric composition i n human plasma, urine and s a l i v a by this method has d e f i n i t e advantages compared to assays based on r e s o l u t i o n of the diastereomers on conventional packed columns. Employing the new technique developed, the pharmacokinetics of i n d i v i d u a l enantiomers of tocainide fo l lowing adminis t ra t ion of the racemic drug have been studied for the f i r s t time i n healthy subjects and i n a pat ient with rena l dys funct ion . The s t e reose lec t ive method was found to be appl icab le to measurement of tocainide enantiomers i n s a l i v a as w e l l . This study has revealed that there i s e n a n t i o s e l e c t i v i t y i n the excret ion of tocainide enantiomers in to human s a l i v a and that the enantiomeric composition of - 205 -the s a l i v a i s d i f f e r e n t from that of plasma or u r i n e . These observations are i n d i c a t i v e of a spec i a l mechanism of excret ion of th i s drug in to the s a l i v a which cannot be explained on the basis of pH of the s a l i v a or pKa of the drug. Tocainide enantiomer d i s p o s i t i o n studies i n a pat ient with renal dysfunct ion revealed that the h a l f - l i v e s of both enantiomers are increased 2 . 5 - f o l d compared to h a l f - l i v e s i n healthy subject s , while the clearance i s decreased by 40%. The f r a c t i o n of the drug removed by d i a l y s i s a l so has been determined. Pre l iminary studies on the i s o l a t i o n and c h a r a c t e r i s a t i o n of toca in ide glucuronides from the urine have revealed d i r e c t mass spec t ra l evidence for the proposed s tructure of tocainide carbamoyl g lucuronide . Ac id or enzyme hydro lys i s s tudies of ur ine have shown the presence of a large excess of a glucuronide derived from the R(-) isomer, suggesting a s t e reose lec t ive g lucuronidat ion pathway. 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