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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 Pharra., B i r l a M.Sc,  I n s t i t u t e of  The U n i v e r s i t y of  PILLAI  Technology & S c i e n c e , 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  in THE FACULTY OF GRADUATE STUDIES ( F a c u l t y of  Pharmaceutical  Sciences)  D i v i s i o n of  Pharmaceutical  Chemistry  We accept t h i s to  the  t h e s i s as  required  THE UNIVERSITY  conforming  standard  OF BRITISH COLUMBIA  July  1984  © Gopalakrishna P i l l a i ,  1984  1967  In presenting this thesis in partial f u l f i l m e n t of the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t 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 a g r e e t h a t , t h e 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 that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or p u b l i c a t i o n of t h i s t h e s i s for financial gain shall n o t be a l l o w e d w i t h o u t my w r i t t e n permission.  Faculty of Pharmaceutical Sciences D e p a r t m e n t o f 22 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 1956 M a i n M a l l V a n c o u v e r , Canada V6T 1Y3  A u g u s t 17,  1984  -  ii  -  ABSTRACT  Tocainide, a structural effective includes racemic  analog of l i d o c a i n e ,  a n t i a r r h y t h m i c agent. an asymmetric c e n t e r  form.  enantiomers  and the drug i s  used  than the S(+)  i n man, the R(-)  isomer  lies  capable  and s e l e c t i v e  required  that  together  in biological fluids.  of  t o c a i n i d e enantiomers  analytical  The main o b j e c t i v e  to study  technique  when present  of t h i s  study was  i n human plasma,  u r i n e and s a l i v a  and to  disposition.  stationary  capillary  phase ( C h i r a s i l - V a l ® )  developed  for  Base-line  r e s o l u t i o n of t h e i r h e p t a f l u o r o b u t y r y l d e r i v a t i v e s  the d i r e c t r e s o l u t i o n of t o c a i n i d e  a c h i e v e d and no i n t e r f e r i n g peaks were observed The i d e n t i t y of the r e s o l v e d  comparison of the r e t e n t i o n t i m e , o p t i c a l tocainide  enantiomers,  to  measurement  chromatographic method u s i n g a f u s e d - s i l i c a  extracts.  is  the simultaneous  column coated w i t h a c h i r a l  saliva  their  for  examine t h e i r s t e r e o s e l e c t i v e A gas  three  distribution,  In order  of r e s o l v i n g the two isomers  develop such a method and to apply i t  tocainide  can r e v e a l whether  i n the process of a b s o r p t i o n ,  a very s e n s i t i v e is  of the  i n the  as an a n t i a r r h y t h m i c agent i n a  metabolism or a combination of these p r o c e s s e s . disposition  tocainide  enantiomer i s  Pharmacokinetic s t u d i e s of enantiomers  the s t e r e o s e l e c t i v i t y  of  clinically  Although the a n t i a r r h y t h m i c p r o p e r t i e s  times more potent  an o r a l l y  The c h e m i c a l s t r u c t u r e  have not been s t u d i e d  mouse model.  is  was  enantiomers.  i n plasma,  was  urine or  peaks was e s t a b l i s h e d  r o t a t i o n and mass s p e c t r a  which were obtained by a  stereospecific  by of  -  synthesis.  iii -  In o r d e r to study s t e r e o s e l e c t i v e d i s p o s i t i o n ,  200 mg of ( ± )  t o c a i n i d e h y d r o c h l o r i d e ( t a b l e t s ) were g i v e n o r a l l y to seven male s u b j e c t s and by i n t r a v e n o u s was withdrawn at  i n f u s i o n to f i v e of them.  predetermined time i n t e r v a l s  Blood (8 mL)  up to 72 hours and u r i n e  was c o l l e c t e d up to 96 hours f o l l o w i n g drug a d m i n i s t r a t i o n . mL) samples were a l s o c o l l e c t e d at  Saliva  (2  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 d a t a and p r e c i s i o n of assay of plasma, saliva  healthy  were determined by t r i p l i c a t e  a n a l y s e s of  six  u r i n e and  concentrations  r a n g i n g from 50 ng to 3000 ng of ( ± ) t o c a i n i d e h y d r o c h l o r i d e along with 1000 ng of  1-aminoacetoxylidide  The r e l a t i v e plasma,  2.6  standard to 7.2%  c o n c e n t r a t i o n that  deviations  (W-49167)  as  f o r u r i n e and 2.1  to 5.9% f o r  c o n c e n t r a t i o n - t i m e d a t a were analysed  hours f o r  the d e x t r o - i s o m e r  hours and 11.7  ± 2.4  L/Kg and 2.52  The plasma  The h a l f - l i v e s  f o l l o w i n g an o r a l d o s e , were 16.3  and 11.9  ± 2.7  hours f o r  the  f o l l o w i n g an intravenous dose were 17.0  hours r e s p e c t i v e l y .  L/Kg f o r  The plasma c l e a r a n c e s  the d e x t r o -  the d e x t r o -  and  were reached at  were absorbed  the same t i m e .  ± were  levo-isomers,  and l e v o - i s o m e r s  F o l l o w i n g o r a l a d m i n i s t r a t i o n , both isomers same r a t e and peak l e v e l s  ± 4  levo-isomer.  The volumes of d i s t r i b u t i o n , (V<j)3, were 2.47 ± 0.49  injection  by a computer program (AUTOAN and  144 ± 28 mL/min and 222 ± 54 mL/min f o r respectively.  for  The lowest  mode of  a two compartment model.  from the plasma d a t a ,  The c o r r e s p o n d i n g h a l f - l i v e s 2.5  saliva.  could be determined by the s p l i t  NONLIN) and were found to f i t  standard.  were i n the range of 4 to 9.9%  was 25 ng of each enantiomer per mL of plasma.  calculated  the i n t e r n a l  ± 0.43 respectively.  r a p i d l y at  the  The area under  -  iv -  the plasma c o n c e n t r a t i o n - t i m e curve f o l l o w i n g o r a l dose was h i g h e r than that  f o l l o w i n g 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 and t h e r e f o r e  the  b i o a v a i l a b i l i t y was 151 ± 43% f o r the d e x t r o - isomer and 167 ± 67% f o r the l e v o - i s o m e r .  B i o a v a i l a b i l i t y calculated  18% f o r the d e x t r o - i s o m e r  and 112 ± 29% f o r  tocainide/(-)  the l e v o - i s o m e r .  enantiomer r a t i o ,  (+)  tocainide,  24 hours and 1.88  at 48 hours f o l l o w i n g an o r a l dose of the  The mean  i n the plasma was 1.52  i n the u r i n e but enantiomer  c o m p o s i t i o n i n the s a l i v a was d i f f e r e n t  i n that  isomers  and 3.68  c o n c e n t r a t i o n r a t i o was 2.06  ± 0.76  for  the l e v o - i s o m e r .  plasma c o n c e n t r a t i o n , ranged from, dextro-isomer  dysfunction  and r = 0.884 to 0.986 f o r  of both the  to a 2.5  the  was decreased  to 0.987 for  the Tocainide  i n a patient with renal The h a l f - l i f e  of  the  the l e v o - i s o m e r was 28.7  increase  levo-isomers, d i s p o s i t i o n of  respectively.  to 6.5  respectively.  hours,  as compared to h e a l t h y  t o c a i n i d e enantiomers  the enantiomer r a t i o  During h e m o d i a l y s i s ,  hours and 5.4  Thus i t  The  dextro-isomer  The plasma c l e a r a n c e was 86 mL/min and 146 mL/min f o r  and l e v o - i s o m e r s ,  half-life  fold  for  the l e v o - i s o m e r .  (serum c r e a t i n i n e = 13.8 mg%).  which corresponded subjects.  ± 0.50  r = 0.910  d e x t r o - i s o m e r was 45 hours and that of  that  the l e v e l s  The c o r r e l a t i o n between s a l i v a and  enantiomer d i s p o s i t i o n was a l s o s t u d i e d  dextro-  the l e v o - i s o m e r l e v e l  were h i g h e r than the c o r r e s p o n d i n g plasma c o n c e n t r a t i o n s .  saliva/plasma  at  racemate.  S i m i l a r enantiomer r a t i o s were observed  was h i g h e r than the d e x t r o - isomer and that  \  from u r i n e data was 109 ±  hours f o r  the  the d e x t r o - and  can be concluded that i n the human i s  the  the  s t e r e o s e l e c t i v e and  i n the plasma i s v a r i a b l e between  subjects.  -  v -  TABLE OF CONTENTS Page ABSTRACT  ii  LIST OF TABLES  xiii  LIST OF FIGURES  xvi  SYMBOLS AND ABBREVIATIONS  xx  ACKNOWLEDGEMENTS  I.  xxii  INTRODUCTION A.  1  Plasma C o n c e n t r a t i o n M o n i t o r i n g of A n t i a r r h y t h m i c Drugs  B.  1  Pharmacokinetics  4  1.  A b s o r p t i o n and Plasma  2.  Intravenous A d m i n i s t r a t i o n  7  3.  Distribution  8  4.  Metabolism and E l i m i n a t i o n  9  5.  Effects  6.  Plasma  7.  Side e f f e c t s  13  8.  Drug I n t e r a c t i o n s  15  9.  Comparison w i t h other A n t i a r r h y t h m i c Drugs  16  10.  Concentrations  of D i s e a s e on P h a r m a c o k i n e t i c s Concentrations  Methods of A n a l y s i s Biological  C.  of T o c a i n i d e  and C l i n i c a l  of T o c a i n i d e  Fluids  effects  7  11 12  in 16  S t e r e o s e l e c t i v e Drug A n a l y s i s  20  1.  Resolution  20  2.  Direct Resolution  of Diastereomers of Enantiomers  21  -  vi  -  Page 3.  R e s o l u t i o n of Enantioraers  by HPLC  4.  S t e r e o s p e c i f i c Radioimmunoassays  23  5.  R e s o l u t i o n of T o c a i n i d e Enantiomers  24  and Drug Accumulation  D.  Renal F a i l u r e  E.  G l u c u r o n i d a t i o n i n Renal F a i l u r e  29  F.  Enantioselectivity  31  i n Drug D i s p o s i t i o n  Enantioselective  Distribution  31  2.  Enantioselective  Metabolism  33  I n v e r s i o n of  C o n f i g u r a t i o n as a M e t a b o l i c  Process G.  Specific  34  Aims of the P r o j e c t  35  EXPERIMENTAL A.  B.  25  1.  3.  II.  22  Materials  39 and Chemicals  39  1.  Column Chromatography  39  2.  T h i n - l a y e r Chromatography  39  3.  High-performance L i q u i d  39  4.  G a s - l i q u i d Chromatography  40  5.  Animal Surgery  40  6.  Miscellaneous  41  Preparation  Chromatography  of Reagents and Stock  Solutions  41  1.  Tocainide Hydrochloride Solution  41  2.  W-49167 H y d r o c h l o r i d e S o l u t i o n  42  3.  a-Bromonaphthalene  42  4.  Monoethylglycinexylidide Hydrochloride S o l u t i o n . . .  42  5.  T o c a i n i d e Base S o l u t i o n  42  6.  N a p h t h o r e s o r c i n o l Reagent  43  Solution  -  vii  -  Page C.  P r e l i m i n a r y S t u d i e s on C a p i l l a r y Column Gas Chromatography of T o c a i n i d e  43  1.  43  Column S e l e c t i o n . . . . 1.1  P r e p a r a t i o n 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) A n a l y s 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  GLC A n a l y s i s of T o c a i n i d e Using C a p i l l a r y Columns Coated w i t h S i l a r 10 C, OV-225 and SP-2330  45  1.3  2.  D.  D e t e r m i n a t i o n of Optimum C o n d i t i o n s Derivatization  3.  Selection  of  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 T o c a i n i d e and I n t e r n a l S t a n d a r d , M o n o e t h y l g l y c i n e x y l i d i d e (MEGX)  Pharmacokinetics 1.  Internal  for 45  Standard  46 Between 46  of T o c a i n i d e i n the Rat  47  Plasma L e v e l Study  47  1.1  Preparation  47  1.2  Surgical  1.3  Drug A d m i n i s t r a t i o n and S e r i a l  of J u g u l a r V e i n Cannula  Implantation  of  the Cannula  48  Blood  Collection 2.  49  Urinary E x c r e t i o n Studies  50  E.  D e t e r m i n a t i o n of C a l i b r a t i o n Curve Data and  F.  P r e c i s i o n of Rat Plasma and U r i n e Assay R e s o l u t i o n of T o c a i n i d e Enantiomers on C h i r a s i l - V a l ® G l a s s C a p i l l a r y Column  G.  S y n t h e s i s of T o c a i n i d e Enantiomers  52  1.  S y n t h e s i s of R ( - ) t o c a i n i d e  hydrochloride  52  2.  S y n t h e s i s of  S(+)tocainide hydrochloride  53  50 t  51  -  viii  -  Page 3.  4.  H.  1.  2.  D e t e r m i n a t i o n of O p t i c a l P u r i t y and I d e n t i t y of R(-) and S(+) T o c a i n i d e by Gas Chromatography/Mass Spectrometry (GCMS)  54  Measurement Enantiomers  55  of  O p t i c a l R o t a t i o n of  Tocainide  D e t e r m i n a t i o n 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 T o c a i n i d e Enantiomers i n Human Plasma and U r i n e  55  I n t e r - and I n t r a - a s s a y V a r i a t i o n s  55  I.  P r e l i m i n a r y Study of S t e r e o s e l e c t i v i t y of D i s p o s i t i o n i n the Humans  J.  1.  2.  K.  2.  L.  M.  56  Comparison of A n a l y t i c a l R e s u l t s Obtained f o r Racemic T o c a i n i d e Using Carbowax 20 M Fuseds i l i c a C a p i l l a r y Column w i t h those of T o c a i n i d e Enantiomers Using C h i r a s i l - V a l ® G l a s s C a p i l l a r y Column  57  D e t e r m i n a t i o n 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  D e t e r m i n a t i o n of Precision 1.  Tocainide  C a l i b r a t i o n Curve Data and 58  Human Plasma and U r i n e 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 I n t e r n a l Standard  58  C o n f i r m a t i o n of the S t r u c t u r e of A c 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  58  The E f f e c t of Sodium Hydroxide Treatment on U r i n e Containing Tocainide Carbamoyl-0-3-D-Glucuronide.....  58  Pharmacokinetics of T o c a i n i d e Enantiomers H e a l t h y Human Subjects  in 60  1.  Intravenous A d m i n i s t r a t i o n of Racemic Hydrochloride  Tocainide  2.  O r a l A d m i n i s t r a t i o n of Racemic Hydrochloride  60  Tocainide 60  -  ix  -  Page N.  1.  Assay of T o c a i n i d e H y d r o c h l o r i d e T a b l e t s  61  2.  Assay of T o c a i n i d e H y d r o c h l o r i d e I n j e c t i o n  61  3.  D e t e r m i n a t i o n of A d s o r p t i o n of T o c a i n i d e by the P l a s t i c  0.  1.  2.  3.  4.  the I n f u s i o n  Set  62  Chromatographic A n a l y s i s of Uremic Plasma E x t r a c 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  Chromatographic A n a l y s i s of Uremic Plasma E x t r a c t s 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  P r e l i m i n a r y Study of T o c a i n i d e Enantiomer D i s p o s i t i o n i n an A n e p h r i c P a t i e n t  63  4.2  Intravenous  A d m i n i s t r a t i o n of  Racemic  Tocainide Hydrochloride  63  Hemodialysis  64  D e t e r m i n a t i o n 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  Q.  62  Chromatographic A n a l y s i s of Uremic Plasma E x t r a c t 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  4.1  P.  Tubing of  Analysis 1.  64  of T o c a i n i d e M e t a b o l i t e s  Isolation  of G l u c u r o n i d e s  i n Human U r i n e  65  from U r i n e by  A d s o r p t i o n on XAD-2 R e s i n Column T h i n - l a y e r Chromatographic S e p a r a t i o n of the Glucuronides P r e p a r a t i v e T h i n - l a y e r Chromatographic I s o l a t i o n of T o c a i n i d e G l u c u r o n i d e s  66  4.  M i c r o b o r e LCMS of T o c a i n i d e G l u c u r o n i d e s  68  5.  I d e n t i f i c a t i o n of Band I b as Carbamoyl-0-3-D-glucuronide  68  2. 3.  5.1  65  66  Tocainide  L i q u i d Chromatographic A n a l y s i s of the Hydantoin D e r i v e d from the G l u c u r o n i d e . . . . . .  68  -  x Page  5.2  5.3  5.4  6.  III.  GC and GCMS A n a l y s i s of the Hydantoin D e r i v e d from the G l u c u r o n i d e s  69  A c i d H y d r o l y s i s of the G l u c u r o n i d e T o c a i n i d e Enantioraers  70  to  Enzyme H y d r o l y s i s of the G l u c u r o n i d e T o c a i n i d e Enantiomers  Gas Chromatography/Mass  Spectrometry  to 70  (GCMS) of  Tocainide Glucuronides  71  6.1  P e r m e t h y l a t i o n of G l u c u r o n i d e s  71  6.1.1  Preparation  of Dry DMSO  71  6.1.2  Preparation  of Dimsylsodium  6.1.3  Carbanion Permethylation  71 72  RESULTS AND DISCUSSION A.  74  G a s - l i q u i d Chromatographic A n a l y s i s of T o c a i n i d e Enantiomers U s i n g 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.  Selection  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 T o c a i n i d e and I n t e r n a l Standard, MEGX  3.  Minimum D e t e c t a b l e Quantity  4.  A p p l i c a t i o n of C a p i l l a r y Gas Chromatography the Assay of Rat Plasma and U r i n e  5.  of S u i t a b l e I n t e r n a l  Pharmacokinetics  Standard Between  83  of Intravenous T o c a i n i d e  for  Resolution  83  in  the Rat B.  82  86 of T o c a i n i d e Enantiomers  1.  Identification  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  3.  T o c a i n i d e Enantiomers i n Human Plasma and U r i n e . . P r e l i m i n a r y Study of S t e r e o s e l e c t i v i t y i n T o c a i n i d e D i s p o s i t i o n i n Man  103  E f f e c t of Sodium Hydroxide Treatment on U r i n e Containing Tocainide-carbamoyl-O-^D-Glucuronide.  Ill  4.  of Resolved  89  Peaks  95 of 98  -  xi  -  Page C.  Pharmacokinetics  i n Man  1.  Pharmacokinetics  of O r a l T o c a i n i d e E n a n t i o m e r s . . .  2.  Pharmacokinetics Enantiomers  of Intravenous  118 118  Tocainide 121  2.1  Plasma C l e a r a n c e  132  2.2  Renal C l e a r a n c e  133  2.3  Volume of D i s t r i b u t i o n  133  3.  T o c a i n i d e Enantiomer L e v e l s  4.  Chromatographic A n a l y s i s of Uremic Plasma E x t r a c 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  Pharmacokinetics of T o c a i n i d e Enantiomers Renal D y s f u n c t i o n and D u r i n g Hemodialysis  149  5.  of Intravenous  134  in  Pharmacokinetics Enantiomers  5.2  D i s p o s i t i o n of Drug G l u c u r o n i d e s i n Renal Failure  157  Hemodialysis  158  5.3.1  6.  i n the U r i n e  5.1  5.3  Analysis  Tocainide  Clearance C a l c u l a t i o n s D u r i n g Hemodialysis  Stereoselective Salivary Enantiomers i n Man 6.1  D.  of T o c a i n i d e Enantiomers  E x c r e t i o n of  of T o c a i n i d e M e t a b o l i t e s  160  Tocainide  T o c a i n i d e Enantiomers i n the S a l i v a P a t i e n t w i t h Renal D y s f u n c t i o n  152  163  of a 172  i n the U r i n e  175  of G l u c u r o n i d e s  177  A n a l y s i s of G l u c u r o n i d e s . . .  178  1.  Gas Chromatographic A n a l y s i s  2.  L i q u i d Chromatographic  3.  Isolation  4.  M i c r o b o r e LCMS of T o c a i n i d e G l u c u r o n i d e s  5.  Identification  of G l u c u r o n i d e s from U r i n e  of Band 1^ as  Carbamoyl-O-3-D-Glucuronide  179 182  Tocainide 184  -  xii  -  Page 6.  Gas Chromatographic/Mass S p e c t r o m e t r i c (GCMS) A n a l y s i s of Permethylated G l u c u r o n i d e s 6.1  6.2  6.3  6.4  CI and EI Mass S p e c t r a Glucuronic Acid  189  of Permethylated 189  CI and EI Mass S p e c t r a of Permethylated p-Ni t r o p h e n o l G l u c u r o n i d e  193  GCMS of Permethylated T o c a i n i d e G l u c u r o n i d e s U s i n g 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  GCMS of Permethylated T o c a i n i d e G l u c u r o n i d e s Using a C h i r a s i l - V a l ® F u s e d - s i l i c a Capillary  Column  201  SUMMARY AND CONCLUSIONS  204  REFERENCES  206  -  xiii  -  LIST OF TABLES  Table  Page  1  Area r a t i o s at  different  split  ratios.  2  C a l i b r a t i o n curve d a t a and p r e c i s i o n of and u r i n e assay  83 rat  plasma 87  3  P h a r m a c o k i n e t i c parameters of i n t r a v e n o u s i n the r a t s  4  Percent drug e x c r e t e d urine  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 t o c a i n i d e enantiomers i n human plasma  101  6  C a l i b r a t i o n d a t a and p r e c i s i o n of assay of enantiomers i n human u r i n e  102  unchanged i n the 24 hour  7  Inter-  8  Plasma c o n c e n t r a t i o n - t i m e dose of 3 mg/kg t o c a i n i d e  9  tocainide  and i n t r a - s a m p l e  variations  rat  88  89  tocainide  i n area r a t i o s  105  \  d a t a f o l l o w i n g an o r a l to two h e a l t h y s u b j e c t s  106  J \  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 u s i n g carbowax 20 M f u s e d - s i l i c a c a p i l l a r y column  . N\$*f .„108-~7 ± ^  10  H a l f - l i v e s of t o c a i n i d e enantiomers i n two h e a l t h y s u b j e c t s  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 u s i n g 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  113  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 urine 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  Plasma c o n c e n t r a t i o n - t i m e d a t a f o l l o w i n g an o r a l dose of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e i n seven h e a l t h y v o l u n t e e r s  119  Pharmacokinetic parameters of t o c a i n i d e f o l l o w i n g an o r a l dose of 200 mg of ( ± ) hydrochloride  120  12  13  14  and the raceraate  107  enantiomers tocainide  -  xiv  -  Page 15  16  17  18  19  20  21  22  23  24  25  26  27  Plasma c o n c e n t r a t i o n - t i m e data f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y v o l u n t e e r s  123  Enantiomer r a t i o , ( + ) T 0 C / ( - ) T 0 C , i n plasma f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y v o l u n t e e r s  124  Pharmacokinetic parameters of t o c a i n i d e enantiomers f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y v o l u n t e e r s . . . .  127  Pharmacokinetic parameters of t o c a i n i d e enantiomers ( c a l c u l a t e d from AUC) f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y v o l u n t e e r s  128  B i o a v a i l a b i l i t y (%) plasma d a t a  129  of t o c a i n i d e enantiomers  Assay of standard t o c a i n i d e and of samples from t a b l e t s and i n j e c t i o n s  from  prepared 131  Assay r e s u l t s before and a f t e r the i n j e c t i o n of t o c a i n i d e h y d r o c h l o r i d e i s passed through the i n f u s i o n set  132  Cumulative e x c r e t i o n of t o c a i n i d e enantiomers i n the u r i n e (%) f o l l o w i n g o r a l and i n t r a v e n o u s dose of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e  135  U r i n a r y e x c r e t i o n d a t a f o r the r a t e p l o t : dosei . v . i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e . . .  140  U r i n e data f o r amount remaining to be e x c r e t e d (ARE) p l o t of S(+) t o c a i n i d e : dose i . v . i n f u s i o n of 200 mg ( ± ) tocainide hydrochloride  141  H a l f - l i v e s of t o c a i n i d e plasma and u r i n e data  142  enantiomers  calculated  from  Enantiomer r a t i o , ( + ) T 0 C / ( - ) T 0 C , i n the plasma and u r i n e f o l l o w i n g an o r a l dose of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e to seven h e a l t h y v o l u n t e e r s  143  C a l i b r a t i o n d a t a and p r e c i s i o n of uremic plasma assay u s i n g dual c a p i l l a r y column (carbowax 20 M and Chirasil-Val®)  151  -  XV  -  Page 28  Plasma c o n c e n t r a t i o n - t i m e data f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t with r e n a l d y s f u n c t i o n  153  Pharmacokinetic parameters of t o c a i n i d e enantiomers f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t with r e n a l d y s f u n c t i o n and d u r i n g h e m o d i a l y s i s  156  30  A r t e r i a l and venous c o n c e n t r a t i o n s t o c a i n i d e and the A-V d i f f e r e n c e  162  31  C a l i b r a t i o n curve d a t a and p r e c i s i o n of assay of saliva  165  T o c a i n i d e enantiomers i n the s a l i v a of h e a l t h y s u b j e c t s f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e  166  Saliva/plasma concentration ratios enantiomers i n h e a l t h y v o l u n t e e r s  167  29  32  33  34  35  36  of  of  the isomers  of  tocainide  Enantiomer r a t i o , ( + ) T 0 C / ( - ) T 0 C , i n plasma, u r i n e and s a l i v a f o l l o w i n g i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to h e a l t h y v o l u n t e e r s  171  Comparison of r a t i o of S(+) volunteers  173  observed and p r e d i c t e d s a l i v a / p l a s m a t o c a i n i d e i n one of the h e a l t h y  T o c a i n i d e enantiomers i n the s a l i v a r e n a l d y s f u n c t i o n and s a l i v a / p l a s m a  of a p a t i e n t ratios  with 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 l u c u r o n i c a c i d i n the CI and EI mass s p e c t r a  194  38  R e l a t i v e i n t e n s i t i e s of fragment ions of permethylated p - n i t r o p h e n o l g l u c u r o n i d e i n the CI and EI mass spectra  196  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 l u c u r o n i d e s of t o c a i n i d e e x t r a c t e d from u r i n e  199  39  -  xvi -  L I S T OF FIGURES Figure  Page  1  Structures  2  Chromatogram of p o l a r i t y m i x t u r e 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  Chromatogram of h e p t a f l u o r o b u t y r y l d e r i v a t i v e of t o c a i n i d e 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  Chromatograms of h e p t a f l u o r o b u t y r y l d e r i v a t i v e of t o c a i n i d e on S i l a r 10 C, 0V-225 and SP-2330 g l a s s c a p i l l a r y columns  79  5  Time-dependence o f d e r i v a t i v e f o r m a t i o n  80  6  S t r u c t u r e o f m o n o e t h y l g l y c i n e x y l i d i d e (MEGX)  81.  7  ECD response o f 3 picograms of t o c a i n i d e on carbowax  3  4  of t o c a i n i d e and l i d o c a i n e  20 M f u s e d - s i l i c a 8  A.  B.  9  capillary column...  Rat plasma p r o f i l e 3 hours a f t e r of t o c a i n i d e h y d r o c h l o r i d e Rat u r i n e c o n t a i n i n g 1.0 internal  Structures  5  standard  84 i n t r a v e n o u s dose 85  ug each of t o c a i n i d e and  (MEGX)  85  of t o c a i n i d e enantiomers  10  Stereoselective  11  S t r u c t u r e of C h i r a s i l - V a l ®  12  Rat u r i n e p r o f i l e 24 hours a f t e r an i n t r a v e n o u s dose of ( ± ) t o c a i n i d e h y d r o c h l o r i d e on C h i r a s i l - V a l ® g l a s s c a p i l l a r y column  94  13  Chromatogram of ( ± ) t o c a i n i d e h e p t a f l u o r o b u t y r a t e C h i r a s i l - V a l ® g l a s s c a p i l l a r y column  96  14  Synthesis  15  T o t a l i o n - c u r r e n t p r o f i l e of h e p t a f l u o r o b u t y r y l d e r i v a t i v e of R(-) t o c a i n i d e  16  events  90  of R(-)  p r i o r to b i o l o g i c a l r e s p o n s e . . . . stationary  phase  93  on  tocainide  EI mass s p e c t r a of h e p t a f l u o r o b u t y r y l d e r i v a t i v e s t o c a i n i d e enantiomers  91  97  99 of 100  -  xvii  -  Page 17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  Plasma, u r i n e and s a l i v a p r o f i l e 24 hours a f t e r o r a l dose of ( ± ) t o c a i n i d e h y d r o c h l o r i d e  an 104  T o c a i n i d e 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  Structures tocainide  110  of  1-aminoacetoxylidide  (W-49167) and  CI mass s p e c t r a of h e p t a f l u o r o b u t y r y l d e r i v a t i v e o f i n t e r n a l s t a n d a r d , W-49167  112  T o c a i n i d e enantiomers: plasma p r o f i l e 24 hours an o r a l dose of the racemate  115  T o c a i n i d e enantiomers: u r i n e p r o f i l e 92 hours an o r a l dose of the racemate  after  after 116  T o c a i n i d e enantiomers: plasma c o n c e n t r a t i o n - t i m e curves a f t e r an o r a l dose of the racemate  122  T o c a i n i d e enantiomers: plasma c o n c e n t r a t i o n - t i m e curves a f t e r an i n t r a v e n o u s i n f u s i o n of ( ± ) t o c a i n i d e . .  126  Cumulative amount of S(+) urine  137  tocainide excreted  i n the  S e m i l o g a r i t h m i c p l o t of e x c r e t i o n r a t e versus time a f t e r an 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 of ( ± ) t o c a i n i d e hydrochloride  138  S e m i l o g a r i t h m i c p l o t of the amount remaining to be e x c r e t e d v e r s u s time (ARE p l o t ) a f t e r an i n t r a v e n o u s dose of ( ± ) t o c a i n i d e h y d r o c h l o r i d e  139  U r i n e p r o f i l e before with 3-glucuronidase  145  (A) and a f t e r  (B)  hydrolysis  GC/ECD p r o f i l e of uremic plasma blank on f u s e d - s i l i c a c a p i l l a r y column  Chirasil-Val® 147  Chromatogram of blank uremic plasma e x t r a c t c o n t a i n i n g 16.8 mg % c r e a t i n i n e 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  GC/ECD p r o f i l e of uremic plasma blank 2 hours a f t e r i n t r a v e n o u s i n f u s i o n of ( ± ) t o c a i n i d e to an a n e p h r i c patient. 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  -  xviii  -  Page 32  33  Log plasma c o n c e n t r a t i o n - t i m e curve f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t w i t h r e n a l d y s f u n c t i o n  155  Log a r t e r i a l plasma 5 hour h e m o d i a l y s i s  161  c o n c e n t r a t i o n - t i m e curve d u r i n g  34  T o c a i n i d e enantiomers: s a l i v a p r o f i l e one hour i n t r a v e n o u s i n f u s i o n of the racemate  35  Log plasma and s a l i v a c o n c e n t r a t i o n s and S(+) t o c a i n i d e enantiomers  36  vs  time f o r  after  168  R(-) 170  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 t o c a i n i d e enantiomers f o l l o w i n g i n t r a v e n o u s i n f u s i o n of the r a c e m a t e . .  174  37  Isolation  181  38  M i c r o b o r e LCMS of  39  M i c r o b o r e LCMS of p - n i t r o p h e n o l g l u c u r o n i d e  40  HPLC of h y d a n t o i n ( s t a n d a r d )  of  tocainide glucuronides tocainide  from a g l u c u r o n i d e of 41  42  43  from human u r i n e . . . .  glucuronides  183 185  (A) and hydantoin d e r i v e d  tocainide  (B)  186  GC of h y d a n t o i n ( s t a n d a r d ) (A) and h y d a n t o i n d e r i v e d from a g l u c u r o n i d e of t o c a i n i d e (B)  187  CI mass s p e c t r a of a h y d a n t o i n d e r i v a t i v e from a g l u c u r o n i d e of t o c a i n i d e  188  obtained  Chromatogram of the h e p t a f l u o r o b u t y r y l d e r i v a t i v e s of t o c a i n i d e enantiomers obtained by a c i d - h y d r o l y s i s of band l b  190  GC/MS/CI of permethylated g l u c u r o n i c 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 p a t t e r n of permethylated g l u c u r o n i c a c i d .  192  46  GC/MS/CI of permethylated p - n i t r o p h e n o l g l u c u r o n i d e . Column: SE-30 f u s e d - s i l i c a c a p i l l a r y  195  44  -  xix  -  Page 47  48  GCMS of permethylated g l u c u r o n i d e s of Column: SE-30 f u s e d - s i l i c a c a p i l l a r y  tocainide.  GC/MS/EI of permethylated g l u c u r o n i d e s of t o c a i n i d e . Column: Chirasil-Val® fused-silica capillary  198  202  -  XX  -  SYMBOLS AND ABBREVIATIONS a  d i s t r i b u t i o n rate  3  disposition  AUC  a r e a under the plasma l e v e l - t i m e  CI  chemical  ionisation  EI  electron  impact  Cl^  plasma  C1 D  renal  (Vd)„  rate  constant constant curve  clearance clearance  volume o f  distribution  P  (Vd)gg  volume of d i s t r i b u t i o n at  Vc  volume of c e n t r a l  Ke^  elimination rate  k  excretion rate  k  e  compartment constant  constant  non-renal excretion rate  nr  steady-state  KQt  dose g i v e n by i n t r a v e n o u s  AUMC  area under the f i r s t  constant infusion  moment of the plasma l e v e l time  cumulative amount excreted amount removed by Clpp C^ C„  dialysis ,  plasma  i n the  dialysis  clearance  concentration i n a r t e r i a l c o n c e n t r a t i o n i n venous  plasma plasma  urine  curve  -  Qp  plasma  xxi  flow r a t e through d i a l y s e r  blood flow r a t e through H  hematocrit  (+)TOC  S(+)  tocainide  (-)TOC  R(-)  tocainide  HFBA  heptafluorobutyric  HPLC  high-performance l i q u i d  GLC  gas-liquid  ECD  e l e c t r o n capture  i.v.  intravenous  MEGX W-49167  -  dialyser  anhydride chromatography  chromatography detector  monoethylglycinexylidide 1-aminoacetoxylidide  TOCG  tocainide  DMSO  dimethylsulfoxide  K12  transport  r a t e constant  from compartment  K i  transport  r a t e constant  from compartment 2 to compartment 1  2  carbamoyl-0-$-D-glucuronide  1 to compartment 2  - xxii  -  ACKNOWLEDGEMENT  The author wishes to acknowledge guidance and s u p e r v i s i o n The author  is  throughout  also grateful  the c o u r s e of t h i s  his  investigation.  to D r . Jim A x e l s o n f o r h i s c o n s t a n t  and h e l p f u l d i s c u s s i o n s and to D r s . for  D r . K e i t h McErlane f o r  support  Frank A b b o t t , Jim Orr and John  t h e i r h e l p and guidance d u r i n g the c o u r s e of t h i s  study.  A word of  thanks go to M r s . Barbara McErlane f o r ' her a s s i s t a n c e d u r i n g the study,  Price  chemical  to Rowland Burt f o r h i s h e l p i n o b t a i n i n g mass s p e c t r a l d a t a ,  David Kwok f o r h i s a s s i s t a n c e i n m e t a b o l i t e i s o l a t i o n tography  and to Wayne Riggs and Ram K a p i l f o r  to  and l i q u i d chroma-  t h e i r h e l p d u r i n g animal  surgery.  The f i n a n c i a l  support p r o v i d e d by U n i v e r s i t y of B r i t i s h Columbia,  A s t r a Pharmaceuticals gratefully  and B . C . H e a l t h Care Research  acknowledged.  Foundation  is  -  1 -  INTRODUCTION  A.  Plasma C o n c e n t r a t i o n M o n i t o r i n g o f A n t i a r r h y t h m i c Drugs  The  sub-optimum use  cause of t h e r a p e u t i c pharmacokinetics  of p o t e n t i a l l y e f f e c t i v e  failure.  drugs  is  otherwise  similar characteristics  management  or adverse r e a c t i o n s  may l e a d  to inadequate  when a standard  dose i s  with therapeutic  administered.  or serum drug c o n c e n t r a t i o n i s ,  therefore,  i n c r e a s i n g l y recommended to guide and a d j u s t drug treatment clinical and  conditions.  i n many h o s p i t a l s .  a n t i a r r h y t h m i c agents i s r a p i d and s p e c i f i c  chromatographic and gas  now w e l l e s t a b l i s h e d  S i m i l a r m o n i t o r i n g of  and s a f e t y of a n t i a r r h y t h m i c  o p t i m i s a t i o n of  therapy i f  fulfilled:  and  a n t i a r r h y t h m i c and/or t o x i c e f f e c t s (2)  A direct  contribute  plasma  to g r e a t e r  i n patients  could  the f o l l o w i n g b a s i c  are  demonstrated.  (1)  In combination w i t h  treatment.  M o n i t o r i n g of a n t i a r r h y t h m i c drugs facilitate  liquid  and continuous ECG r e c o r d i n g s ,  would s u b s t a n t i a l l y  out  i n t r o d u c t i o n of  high-performance  chromatographic methods.  testing  and c a r r i e d  important  now f e a s i b l e w i t h the recent  c o n c e n t r a t i o n measurements efficacy  is  enzyme immunoassays,  electrophysiological  i n various  T h e r a p e u t i c m o n i t o r i n g of p h e n y t o i n , t h e o p h y l l i n e  aminoglycoside a n t i b i o t i c s  routinely  frequent  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  and pharmacodynamic responses i n p a t i e n t s  D e t e r m i n a t i o n of plasma  a  r e l a t i o n s h i p between plasma  requirements concentration  of the drug can be  Upper and lower l i m i t s of the  greatly  therapeutic  -  2 -  c o n c e n t r a t i o n range are w e l l d e f i n e d . at  (3)  Plasma c o n c e n t r a t i o n  the recommended dosage are h i g h l y v a r i a b l e .  studies indicate  that p r e s e n t l y a v a i l a b l e  Numerous  clinical  a n t i a r r h y t h m i c drugs  these formal reasons for plasma c o n c e n t r a t i o n m o n i t o r i n g . influence  of drug l e v e l s  q u i n i d i n e was f i r s t  on a n t i a r r h y t h m i c e f f i c a c y  l i d o c a i n e ( G i a n n e l l y et a l . ,  Klein,  1971), m e x i l e t i n e  disopyramide et a l . ,  1978;  flecainide et a l . , 1981)  (Niarchose,  Woosley e t a l . ,  (Somani,  1980;  doses of  is  often d i f f i c u l t  f u n c t i o n may i n f l u e n c e these a g e n t s .  It  is  such as  therefore  the d e s i r e d obvious  1977;  al.,  reported  Talbot et a l . , 1976;  1977;  is  1981),  Winkle  l o r c a i n i d e (Meinertz  1980;  Johnston e t  Greene et a l . ,  s i n c e age,  cardiac,  al.,  1978).  therapeutic  relatively small.  the a b s o r p t i o n ,  and  Dosage  hepatic  or r e n a l  w i t h enzyme-inducing  p h e n y t o i n , p h e n o b a r b i t a l or r i f a m p i c i n may a l s o to p r e d i c t whether a g i v e n dose w i l l  plasma c o n c e n t r a t i o n i n i n d i v i d u a l p a t i e n t s .  indications  occur. achieve  Despite  there are no c o n t r o l l e d s t u d i e s a v a i l a b l e  that a n t i a r r h y t h m i c treatment  toxic  d i s t r i b u t i o n and e l i m i n a t i o n of  In a d d i t i o n , drug i n t e r a c t i o n s  difficult  1973),  Ryans and K e r l i n e r , 1979),  the margin between  the a n t i a r r h y t h m i c drugs  adaptation  agents,  1973,  1979), v e r a p a m i l (Eischelbaum et a l . ,  With a few e x c e p t i o n s ,  Subsequently,  ranges were a l s o  t o c a i n i d e (Winkle e t a l . ,  Anderson e t a l . ,  and a p r i n d i n e ( Z i p e s e t  A direct  1967), procainamide (Koch-Weser and  (Campbell, et a l . , 1976),  fulfill  and t o x i c i t y of  r e p o r t e d by Sokolow and Edgar (1950).  the e x i s t a n c e of r e l a t i v e l y narrow t h e r a p e u t i c for  profiles  to prove  guided by drug l e v e l m o n i t o r i n g i s  -  distinctly clinical the  superior  judgment  to a c a r e f u l l y  alone.  a therapeutic  drug l e v e l s following  would  al.,  heart  improve (1)  failure  dangerous v e n t r i c u l a r  the use  is  after  alterations. influence  immunoassay,  lignocaine  to suppress or  of  the degree of  concentrations  of l i d o c a i n e l e v e l s  of h i g h dose p r o p h y l a c t i c l i d o c a i n e  e p i s o d e s of v e n t r i c u l a r  also  administration  proposed  i n patients with  i n two ways.  to i n d i v i d u a l increase  t a c h y c a r d i a might be aided  c o n c e n t r a t i o n measurement  pharmacokinetic  would a l s o  to prevent primary v e n t r i c u l a r  term a n t i a r r h y t h m i c treatment  clinical  are measurable w i t h i n a few  Routine determinations  Long  In  With the r e c e n t l y i n t r o d u c e d enzyme  needs.  (1974)  the  prevent  v a s o a c t i v e drugs can  and i n f u s i o n r a t e s can be adjusted a c c o r d i n g  by L i e e t a l . ,  of  to p a t i e n t s w i t h  minutes  the s a f e t y  that knowledge  acute m y o c a r d i a l i n f a r c t i o n .  Concomitantly a d m i n i s t e r e d  lignocaine requirements.  the is  often d i m i n i s h e d but the  do not allow an e s t i m a t i o n  and  range i t  of a n t i a r r h y t h m i c drugs i n  and/or hypotension  arrhythmias  observed  effective.  Lidocaine administration  such cases l i g n o c a i n e c l e a r a n c e findings  be  at  patient.  therapeutic  (1983) have r e c e n t l y i n d i c a t e d  situations:  congestive  response i s  above the expected  that c h r o n i c therapy w i l l  F o l l a t h et  c o n c e n t r a t i o n obtained  may a l l o w the c l i n i c i a n to  or p h a r m a c o l o g i c a l  concentration is well  unlikely  testing  drug regimen based on  c o n c e n t r a t i o n for an i n d i v i d u a l  However, i f no c l i n i c a l plasma  selected  Concurrent plasma  time of e l e c t r o p h y s i o l o g i c a l  identify  3 -  Firstly,  fibrillation.  (2)  life-threatening by plasma  the d e t e r m i n a t i o n  of  -  individual  4 -  optimum c o n c e n t r a t i o n range d u r i n g the i n i t i a l  term drug t e s t i n g treatment,  it  can be e s t a b l i s h e d .  In j u d g i n g  the adequacy  should be decided whether one i n t e n d s  types of P r e m a t u r e V e n t r i c u l a r C o n t r a c t i o n s e p i s o d e s of v e n t r i c u l a r  tachycardia.  demonstrated  PVC s u p p r e s s i o n  that  total  phase o f  Meyerberg et requires  al.  (1981)  higher  tachycardia.  concentrations  Secondly,  have  plasma  patient  adherence  to the p r e s c r i b e d  r i s k of  treatment  failure  requirements in  t h i s way.  p e r i o d i c measurements of  d u r i n g f o l l o w - u p p r o v i d e an o b j e c t i v e  by drug i n t e r a c t i o n s (3)  Another obvious  B.  but l e s s  frequent  assess the dose  detected  i n d i c a t i o n for  the n e c e s s i t y  i n p a t i e n t s w i t h r e n a l or h e p a t i c  of dosage  failure.  Pharmacokinetics o f Tocainide  new a n t i a r r h y t h m i c drug which i s exhibits effects. oral  al.,  reduce  S i m i l a r l y , altered  T o c a i n i d e (2-aminopropionq-2'6'=xylidide,  of  plasma  or other d i s e a s e s can a l s o be  a n t i a r r h y t h m i c drug l e v e l m o n i t o r i n g i s adaptation  sustained  c r i t e r i o n to  drug regimen and thus  by n o n - c o m p l i a n c e .  all  or o n l y  c o n c e n t r a t i o n of the a n t i a r r h y t h m i c agent than e l i m i n a t i o n of ventricular  of  to suppress  (PVCs) c o m p l e t e l y ,  short  similar  electro-physiological,  In c o n t r a s t  administration,  11-13  structurally  to l i d o c a i n e ,  related  1) i s  a  and  hemodynamic and a n t i a r r h y t h m i c  however, i t  is  w e l l absorbed  ( L a l k a et  al.,  1976;  although the v a r i a b i l i t y was g r e a t e r  after half-life  Graffner  In p a t i e n t s w i t h m y o c a r d i a l i n f a r c t i o n , the mean  was a l s o 13 h o u r s ,  relatively  to l i d o c a i n e  has complete b i o a v a i l a b i l i t y and a plasma  hours i n h e a l t h y v o l u n t e e r s  1980).  fig.  et  half-life  than that  of  -5-  FIGURE 1  LIDOCAINE  -  healthy volunteers l e s s potent  (Winkle et a l . ,  than l i d o c a i n e i n i t s  a primary amine i n c o n t r a s t c o n s e q u e n t l y the free  6 -  1976;  R o n f e l d , 1980).  d i r e c t cardiac  to the t e r t i a r y  base i s  effects.  1980).  Tocainide i s  sparingly  soluble  i n water.  suppressing  al.,  a b a s i c drug (pka = 7.7)  al.,  C o l t a r t et a l . ,  1980; al.,  1973;  Roden et 1979;  1981;  ventricular  has been shown to be e f f e c t i v e  in  1974;  1978;  Moore et  Young et a l . ,  Sonnhag,  1980;  Ryden et  al.,  1978;  Winkle et  al.,  1981;  trials  Waleffe  w i t h o r a l t o c a i n i d e g i v e n to p a t i e n t s following a myocardial  et  60% of the p a t i e n t s  with  infarction),  than 75% s u p p r e s s i o n  (Esterbrooks  of t o c a i n i d e c o n t a i n s  et a l . ,  of  1983;  an asymmetric  center  used c l i n i c a l l y i n the racemic form, c o n t a i n i n g equal  of each enantiomer.  enantiomer i s  three  P h a r m a c o l o g i c a l s t u d i e s have shown that  times more potent  than the S(+)  an a n t i a r r h y t h m i c agent i n a mouse model (Byrnes et a l . ,  smaller  al.,  1979).  and the drug i s  difference  Duce et  1981).  The chemical s t r u c t u r e  quantities  al.,  and man ( A l m o t r e f i  1980;  1200 to 2400 mg d a i l y produced g r e a t e r  al.,  the R(-)  et  al.,  e c t o p i c beats ( o f t e n  e c t o p i c beats i n about Ryan et  et  Koransky et a l . ,  In a number of  doses of  It  Schnittger  al.,  is  (Graffner  v e n t r i c u l a r arrhythmias i n b o t h animals 1980;  which  system  The more r e a d i l y s o l u b l e h y d r o c h l o r i d e s a l t  1976).  and Baker,  is  amine, l i d o c a i n e , and  can be a d m i n i s t e r e d by the o r a l or i n t r a v e n o u s r o u t e L a l k a , et  is  Tocainide  l e s s l i p o p h i l i c i n an o c t a n o l / w a t e r  (Ronfeld,  1980;  Tocainide  isomer  1979).  as  The  i n the a n t i a r r h y t h m i c a c t i v i t y between the two enantiomers  i n coronary l i g a t e d  dogs (Byrnes et a l . ,  1979).  is  -  Tocainide is activity,  1.  indicated for  7 -  the treatment  of v e n t r i c u l a r  ectopic  u s i n g a dose of 400 mg every 8 h .  A b s o r p t i o n and Plasma C o n c e n t r a t i o n s F o l l o w i n g o r a l a d m i n i s t r a t i o n of t o c a i n i d e h y d r o c h l o r i d e to  healthy volunteers, approaches  100%.  a b s o r p t i o n occurs  r a p i d l y and b i o a v a i l a b i l i t y  The time to reach peak plasma l e v e l s  dose was approximately w i t h food decreased  1 h in fasting  the peak l e v e l s  studies i n patients  r e c e i v e d 400 mg of  tocainide  three  and delayed  the r a t e of  ( L a l k a et  Similar  were obtained  Winkle et  al.,  1976).  9-10  2.  1976).  in patients  et a l . ,  with frequent  times d a i l y ( M e f f i n  When the dose was i n c r e a s e d  d a i l y i n these p a t i e n t s ,  al.,  absorption,  times a day r e c o r d e d mean plasma  of between 5 and 7 ug/mL ( G r a f f n e r  e c t o p i c beats g i v e n 400 mg three  single  w i t h m y o c a r d i a l i n f a r c t i o n who had  concentrations levels  a  s u b j e c t s , while a d m i n i s t r a t i o n  but the b i o a v a i l a b i l i t y was not a f f e c t e d Several  after  1980).  ventricular  et a l . ,  1977a;  to 600 mg three  the mean plasma c o n c e n t r a t i o n s  rose to  times  about  ug/mL.  Intravenous After  be d e s c r i b e d 1980;  Administration  intravenous  the plasma p r o f i l e of t o c a i n i d e  i n most cases by a b i e x p o n e n t i a l e q u a t i o n ( G r a f f n e r  L a l k a et a l . ,  minutes.  infusion,  1976)  et  w i t h a r a p i d d i s t r i b u t i o n phase o f about  I n f u s i o n of 300 mg over 30 minutes i n h e a l t h y  produced peak plasma l e v e l s  of a p p r o x i m a t e l y  2.5  can al., 10  subjects  Ug/mL ( L a l k a et  al.,  -  1976).  In p a t i e n t s  w i t h m y o c a r d i a l ischaeraia,  t o c a i n i d e over 30 minutes r e s u l t e d 1980).  8 -  In a study of 7 p a t i e n t s  i n f u s i o n of 500 mg of  i n plasma l e v e l s  of  11 ug/mL  (Sutton,  w i t h acute m y o c a r d i a l i n f a r c t i o n g i v e n  a combination of 750 mg i n t r a v e n o u s l y , d i r e c t l y followed by 800 mg orally,  and subsequently  plasma l e v e l s  400 mg o r a l l y three  were a t t a i n e d  i n most p a t i e n t s  minutes and remained w i t h i n the t h e r a p e u t i c p e r i o d of o b s e r v a t i o n et  al.,(1982)  minutes levels  administered  to 14 p a t i e n t s ranged  et a l . ,  ug/mL) w i t h i n 15  range throughout the 48 hr  1980).  S i m i l a r l y , when Baxter  500 mg of t o c a i n i d e i n t r a v e n o u s l y over 30  to 19.6  plasma  ug/mL f o l l o w i n g i n f u s i o n , w i t h a mean  ug/mL.  Distribution T o c a i n i d e has been shown to enter  and  (4-10  therapeutic  with suspected m y o c a r d i a l i n f a r c t i o n ,  from 2.4  v a l u e of around 5.8  3.  (Graffner  times d a i l y ,  sheep.  Peak c e r e b r o s p i n a l  1 hour a f t e r  intravenous  fluid  the c e r e b r o s p i n a l  levels  i n f u s i o n of 5 mg/kg/min f o r  r a t i o s between the plasma and c e r e b r o s p i n a l be 1.2  to 1.6  the s t a r t  and 1.1  concentrate  Shortly after  15 m i n u t e s .  levels  The  were found  respectively,  intravenous  of dogs  approximately  to  after  and o r a l  t o c a i n i d e i n m i c e , the drug was seen  high concentrations  N e a r l y a l l of  apart  arteries  of ( 3 H )  fluid  60 and 140 m i n u t e s ,  i n the k i d n e y , g a s t r i c mucosa,  hours l a t e r ,  hours,  1.5,  of the i n f u s i o n .  administrations  arteries.  to  were achieved  fluid  to  and the c h o r o i d p l e x u s .  were found i n the w a l l  Four  of the major  the r a d i o a c t i v i t y had disappeared  after  48  from small amounts i n the kidney and the w a l l s of the major  (Holmes et  al.,  1983).  -  In man, a f t e r half-life and  of  i n f u s i o n of 100 mg of  1980).  found  of 2.9  distribution  and 3.2  patients  of  tocainide.  Binding data,  L/kg, respectively,  for  (1980)  reported  the apparent volume of  g i v e n a 100 mg t o c a i n i d e  i n f u s i o n and  (1982) r e p o r t e d a t o c a i n i d e f r e e and from 0.8  (who had h i g h c o n c e n t r a t i o n s of  consistent  of 750 mg  f r a c t i o n of to 0.9  of  <=^-acid g l y c o p r o t e i n ) .  w i t h the above s t u d y , were r e p o r t e d by Sedman The  t o t a l unbound t o c a i n i d e ranged from about 85 to 90%.  B i n d i n g appeared  to be independent of serum t o c a i n i d e  the range of 4-12  b i n d i n g of  0.78  in 4  (1982) u s i n g serum samples from f i v e h e a l t h y v o l u n t e e r s .  percentage  1982).  G r a f f n e r et a l .  i n sera from 10 normal v o l u n t e e r s  trauma p a t i e n t s  ug/mL.  concentrations  The p o s s i b i l i t y of a d i f f e r e n c e  in  t o c a i n i d e enantiomers has a l s o been examined (Sedman e t  Although b i n d i n g of  than that of R(-)  isomer,  the S(+)  enantioraer was s l i g h t l y  the d i f f e r e n c e  would be of l i t t l e  clinical  M e t a b o l i s m and E l i m i n a t i o n E l v i n et a l .  (1980b) s t u d i e d  the metabolism of  al.,  greater  significance.  4.  et  steady s t a t e was  w i t h acute m y o c a r d i a l i n f a r c t i o n , g i v e n i n f u s i o n s  E l v i n et a l . ,  within  1976).  i n healthy subjects,  in  et a l .  estimated  L / k g on average i n h e a l t h y s u b j e c t s r e c e i v i n g 300 mg  intravenously (Lalka et a l . ,  to 0.96  the  w i t h acute m y o c a r d i a l i n f a r c t i o n ( G r a f f n e r  The apparent volume of d i s t r i b u t i o n at  to be 1.46  values  tocainide,  the d i s t r i b u t i o n phase was 7 minutes i n h e a l t h y s u b j e c t s  13 minutes i n p a t i e n t s  al.,  9 -  tocainide in  humans and r e p o r t e d a novel b i o t r a n s f o r m a t i o n pathway f o r a primary  -  amine,  i n v o l v i n g the formation of  tocainide.  10 -  the g l u c u r o n i d e of  N-carboxy  T h i s g l u c u r o n i d e , u n l i k e any p r e v i o u s l y r e p o r t e d ,  is  thought  to undergo c y c l i z a t i o n to y i e l d a h y d a n t o i n - t y p e compound. Approximately 30% of urine,  together  72 h ( E l v i n  et  tocainide,  1980a).  i n the body.  metabolite  dependent  this  metabolite  compound, as i n animals tocainide  it  is  seems u n l i k e l y  to the b i o l o g i c a l a c t i v i t y  Meffin  1977;  of the  The e l i m i n a t i o n h a l f - l i f e  and o r a l routes  groups ( B e a t t i e et  Woosley et  al.,  degradation  of  al.,  1977;  Graffner  effect  of  following  to h e a l t h y  1978;  that  parent  pharmacological  These v a l u e s were obtained  patient  al.,  potential  It  and to v a r i o u s  et  the  on f u r t h e r metabolism.  by the i n t r a v e n o u s  al.,  has  lactoxylidide  1980).  a  deamination product of  the  al.,  12 to 15 h .  within  (1980) a l s o i d e n t i f i e d  of 26 h and t h e r e f o r e  administration  et  unchanged d r u g ,  been found to have n e g l i g i b l e  (Ronfeld et  form from  The e l i m i n a t i o n of  contributes has  al.,  an o x i d a t i v e  a half-life  to accumulate  the dose as  Ronfeld et  lactoxylidide,  which has  is  been recovered i n t h i s  w i t h 39 to 52% of al.,  g l u c u r o n i d e of  the dose has  subjects  K l e i n et et  al.,  al.,  1980;  1980; Lalka  1976). Since hepatic  elimination,  the e f f e c t s  of h e p a t i c  tocainide  Several  elimination h a l f - l i f e proportion excreted enzyme-inducer,  as  a major  enzyme i n d u c e r s  the r a t e of drug e l i m i n a t i o n i n both animals investigated.  is  and i n h i b i t o r s on  and humans have  s t u d i e s i n r a t s have demonstrated and area under the c u r v e , unchanged drug a f t e r  phenobarbital  route of  (Venkataramanan  a decreased  as w e l l as  pretreatment  been  a lowered  w i t h the  and A x e l s o n ,  1980a,b  -  Bennett e t a l . ,  1980,  1981).  On the other hand,  SKF-525A brought about an i n c r e a s e p r o p o r t i o n of i n t a c t  11 -  the enzyme i n h i b i t o r  in elimination h a l f - l i f e  drug i n the u r i n e and a decrease i n  (Venkataramanan and A x e l s o n , 1 9 8 0 a , b ) .  and  clearance  However, i n the only  study done i n man, there was no evidence of such a l t e r a t i o n s disposition effect  tocainide.  of p r e - t r e a t m e n t  substrates It  of  for  T h u s , E l v i n et a l . ,  enzymes,  was found that p h e n o b a r b i t a l  (in  tocainide  (1980a) examined  the  to m e t a b o l i t e s  excreted  competitive  salicylamide  and  clofibrate.  range of  15-40  change i n blood c o n c e n t r a t i o n s  or the e l i m i n a t i o n r a t e c o n s t a n t .  the o v e r a l l p a t t e r n of r e c o v e r y of  The p r o p o r t i o n s  i n u r i n e were not a l t e r e d . t o c a i n i d e and i t s  u r i n e were not changed by s a l i c y l a m i d e  5.  the  the t h e r a p e u t i c  ug/mL) d i d not induce any s i g n i f i c a n t time p r o f i l e  in  w i t h p h e n o b a r b i t a l and the  the m e t a b o l i s i n g  similar  or  vs  of  Similarly,  glucuronide i n  clofibrate.  E f f e c t s o f D i s e a s e on P h a r m a c o k i n e t i c s Weigers e t a l . ,  patients failure  with renal dysfunction. (creatinine  half-life  was  h) and the longest addition  (1983) s t u d i e d  clearance  significantly  total  half-life  (42.7  prolonged  three were  the r e m a i n i n g p a t i e n t s  than 5 mL/min) the  of  this  tocainide  ranged  from 35  to 94 mL/min.  to 42.7 The  with c i r r h o s i s  had h a l f - l i v e s  longer  the enzyme i n h i b i t o r , a l l o p u r i n o l . group,  i n 15  plasma  to about 27 h (range 16.6  Three p a t i e n t s  taking  of  with end-stage r e n a l  h) was found i n one p a t i e n t  to r e n a l f a i l u r e .  30 h and a l l  In 9 p a t i e n t s  of l e s s  plasma c l e a r a n c e  the d i s p o s i t i o n  the h a l f - l i f e  of  tocainide  in  than In ranged  12 -  -  from 16.6  to 27.5  h (22.3  ± 4.8  h) and the t o t a l  52 to 94 mL/min (68 ± 18 mL/min). function  (creatinine  ranged from 13.2  clearance,  to 22 h (19.2  In s i x  of  decreased  to 8.5  h  the t o c a i n i d e  clearance  During the 4 h of d i a l y s i s  of  al.,  1976;  lidocaine,  G r a f f n e r et  an analog of  uremia (Thomson et  al.,  al.,  tocainide is 1973).  the e l i m i n a t i o n of t o c a i n i d e dysfunction  and that  1980).  is  the apparent  volume of (Lalka  The d i s t r i b u t i o n volume of a l s o unchanged i n p a t i e n t s  From these f i n d i n g s  it  impaired i n patients  the dosage of  the  to removal of 25%  d i s t r i b u t i o n were s i m i l a r to those r e p o r t e d i n h e a l t h y s u b j e c t s et  from  half-life  plasma  which corresponds  Estimates  clearance  with impaired r e n a l  ± 4.0 h) and the t o t a l  ± 4.6  the drug from the body.  patients  10-55 mL/min),  from 72 to 106 mL/min (90 ± 19 mL/min). half-life  plasma  is  with  concluded  that  with renal  t o c a i n i d e must be a d j u s t e d f o r  these  patients.  6.  Plasma C o n c e n t r a t i o n s and C l i n i c a l Studies  relating  t o c a i n i d e plasma  a c t i v i t y vary i n t h e i r r e s u l t s , to 10 ug/mL as  1976;  Winkle et a l . ,  were suppressed by 20% i n p a t i e n t s w i t h plasma increase mg who  concentrations  i n suppression  levels  are c o n s i d e r e d 1976).  of 6 ug/mL and above,  was seen i n p a t i e n t s  to t o c a i n i d e  therapy,  range of 4  acceptable  Ventricular ectopic  taking tocainide  every 8 h w i t h plasma c o n c e n t r a t i o n of 8.5 responded  to a n t i a r r h y t h m i c  but g e n e r a l l y a t h e r a p e u t i c  the h y d r o c h l o r i d e s a l t  (McDevitt et a l . ,  Effects  beats  (400 mg every 8 h)  w h i l e only a  slight  g i v e n a h i g h e r dose of ug/mL.  there was a c l e a r  For the  600  patients  relationship  -  between percentage concentration  13 -  r e d u c t i o n of v e n t r i c u l a r e c t o p i c beats  (Winkle et  al.,  1976;  Meffin  side effects  had serum c o n c e n t r a t i o n s  al.,  Winkle et  of  1976).  al.,  t o c a i n i d e i n 17 p a t i e n t s  steady-state concentrations In  a double b l i n d  (1977) observed concentrations al.,  levels  tocainide  Patients  between 6.9  the long term  and 10.2  effective  of 4.1  to 9.8  and 5-15  et  efficacy  c o n t r o l of  ug/mL.  study i n 12 c a s e s , Woosley et  to 12 ug/mL.  with  between 7 and 16 pg/mL (Winkle  and o b t a i n e d a s a t i s f a c t o r y  cross-over  concentrations  (mean 11.3  steady s t a t e  In more r e c e n t  al.,  tocainide  p a p e r s , Kuck et  ug/mL r e s p e c t i v e l y .  tocainide  Toxic  i n Ryan's s e r i e s v a r i e d between 7 and 18 ug/mL  ug/mL).  Side E f f e c t s The  emergency use  tocainide available intractable  program i n the U . S . A .  f o r use  i n the treatment  (Horn et  of  v e n t r i c u l a r arrhythmias i n p a t i e n t s  unable to take approved a n t i a r r h y t h m i c d r u g s . o p p o r t u n i t y to e v a l u a t e 41.5  1977).  (1979) and Ryan and K a r l i n e r , (1979) r e p o r t e d e f f e c t i v e  plasma  7.  al.,  (1978) a l s o e v a l u a t e d  therapeutically from 3.5  et  and plasma  the s a f e t y  months i n 369 s e r i o u s l y  adverse e f f e c t s lightheadedness, shortness  are nausea, tremors,  unresponsive  or  of up to  dizziness,  nervousness,  i n nature.  to,  The most commonly r e p o r t e d  The m a j o r i t y of  and g a s t r o i n t e s t i n a l  made  p r o v i d e d an  for periods  vomiting, anorexia,  confusion,  1980)  life-threatening  This  tocainide  i l l patients.  of b r e a t h and r a s h .  neurological  of  al.,  palpitations,  side effects  are  The n e u r o l o g i c a l  -  experiences  appear to be r e l a t e d  r e l a t i o n to peak blood l e v e l s . of  to dosage,  withdrawals).  effects  liver  enzymes.  (16%).  tocainide associated hepatitis  (1981) r e p o r t e d  fibrillation  after  al.,  (1982) r e p o r t e d  Both p a t i e n t s  such an e f f e c t f o r 48 h .  clinical  (1982) diagnosed  were of  E n g l e r and LeWinter  disease with  r a d i o l o g i c a l and pulmonary f u n c t i o n changes  improvement i n both c a s e s .  a case of c h r o n i c d i f f u s e  tocainide  Perlow et  pneumonitis w i t h  Withdrawal o f  Brande et  interstitial  and s t a r t i n g  corticosteroids  B r a d y c a r d i a has been r e p o r t e d  al.,  pneumonitis i n a  t a k i n g t o c a i n i d e 400 mg 3 times d a i l y f o r 2 months.  much improved.  pulmonary  i n 12 out of 76  6 months' use of 400 mg t o c a i n i d e 4 times d a i l y .  stopping  after  antiarrhythmic  d i s c o n t i n u a t i o n of t h e r a p y .  (1981) r e p o r t e d two cases of i n t e r s t i t i a l  patient  5 h  r e c u r r e n t v e n t r i c u l a r t a c h y c a r d i a but v e n t r i c u l a r  t o c a i n i d e brought about  after  by v a r i o u s  had severe u n d e r l y i n g heart  d i d not occur a f t e r  characteristic  elevated  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  life-threatening  al.,  In a study of a g g r a v a t i o n  g i v e n 1200 to 2400 mg t o c a i n i d e  tocainide.  and one due to  developed i n t e r s t i t i a l  arrhythmias known to be caused  V a l e b i t et  patients  the  Acute pulmonary edema developed i n one p a t i e n t  r e p o r t e d by L e v i n and Fox ,(1982).  drugs,  termination  N e u r o l o g i c a l and  one because of h e p a t i t i s  Two cases of p o s s i b l e  ventricular  causing  d i s c o n t i n u e d t o c a i n i d e because of  drug a d m i n i s t r a t i o n and another p a t i e n t edema.  possible  were the primary causes of withdrawal (80% o f  Four p a t i e n t s  development of a r a s h ,  suggesting a  Adverse experiences  therapy o c c u r r e d i n 60 p a t i e n t s  gastrointestinal  14 -  One week  the p a t i e n t  in association  with  was  -  intravenous  tocainide  in 3 patients  15 -  ( N y q u i s t et a l . ,  1980;  Sutton,  1980).  8.  Drug I n t e r a c t i o n s The  effect  of pretreatment  w i t h an enzyme i n d u c e r  like  p h e n o b a r b i t a l or c o - a d m i n i s t r a t i o n of a c o m p e t i t i v e s u b s t r a t e f o r metabolising influence  and t o c a i n i d e ,  from sinus  little  such as  salicylamide  the e l i m i n a t i o n of  Metoprolol free  system  tocainide  atrioventricular  with  'Sick-sinus'  1983).  alone.  propranolol. tocainide  difference  1980;  i n response to t o c a i n i d e  compared w i t h those p a t i e n t s serum d i g o x i n  Over a p e r i o d of  16 months the p a t i e n t  from 600 to 2400 mg d a i l y .  with p r o p r a n o l o l f o r u n s t a b l e  given  with  t o c a i n i d e and  was  taking  During t h i s  angina on two  time  occasions.  episodes of severe p a r a n o i a and c o n f u s i o n which  on withdrawl of p r o p r a n o l o l .  or  concentrations  (1982) r e p o r t e d a p a t i e n t  f o l l o w i n g concomitant a d m i n i s t r a t i o n of  Each time he s u f f e r e d subsided  of m y o c a r d i a l  and should be avoided (Ikram,  Rubino and Jackson  i n doses ranging  he was t r e a t e d  patients  syndrome and impaired  T o c a i n i d e d i d not a f f e c t  1979).  severe p a r a n o i a  1980a).  Concurrent a d m i n i s t r a t i o n of d i g o x i n and  produced no s i g n i f i c a n t  (Ryan et a l . ,  to  or hemodynamic parameters.  c o n d u c t i o n , the small d e p r e s s i o n  i n c i d e n c e of adverse e f f e c t s  tocainide  al.,  s i n g l y or i n combination g i v e n to  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  altered  shown not  node d i s e a s e or impaired a t r i o v e n t r i c u l a r c o n d u c t i o n had  However, i n p a t i e n t s  tocainide  has  i n humans ( E l v i n et  i n f l u e n c e on e l e c t r o p h y s i o l o g i c a l  Renard et a l . ,  or c l o f i b r a t e ,  the  -  9.  16 -  Comparison w i t h O t h e r A n t i a r r h y t h m i c Drugs Tocainide,  be at l e a s t as  g i v e n both i n t r a v e n o u s l y and o r a l l y has been found  effective  as  procainamide i n s u p p r e s s i o n  arrhythmias (Agnew and W h i t e l o c k ,  1980;  a study on the comparative e f f e c t s  of  Wasenmiller and Aronow (1980) ventricular  ectopic beats,  treated  patients  dose of  found a g r e a t e r  efficacy  In t h i s  ectopic  with  The comparative e f f i c a c y  gas-liquid  tocainide  1-aminoacetoxylidide  study  t o c a i n i d e was of  comparable  than 75% r e d u c t i o n i n  the d e t e r m i n a t i o n of  The f i r s t  Fluids  t o c a i n i d e have  to a t r i f l u o r o a c e t a m i d e  as i n t e r n a l standard  t o c a i n i d e and the i n t e r n a l standard with r e t e n t i o n times  of 4.0  for  liquid  GLC method r e p o r t e d c o n s i s t e d  of  d e r i v a t i v e along with  and a n a l y s i s  on 5% OV-17 packed column ( M c D e v i t t et  c a l i b r a t i o n curves  of a lower  a g a i n s t q u i n i d i n e was s t u d i e d by  chromatographic and high-performance  chromatographic t e c h n i q u e s .  derivatives  treated  beats.  A n a l y t i c a l methods f o r  c o n v e r s i o n of  i n the  than i n the group  Methods o f A n a l y s i s o f T o c a i n i d e i n B i o l o g i c a l  included  frequent  over placebo l e v e l s )  to q u i n i d i n e i n a c h i e v i n g g r e a t e r  ventricular  10.  (1982).  In a r e p o r t of  response r a t e (based on 75%  (46% response r a t e )  t o c a i n i d e (600 mg twice d a i l y )  Morganroth e t a l . ,  1980).  s t u d y i n g 22 p a t i e n t s  with- t o c a i n i d e (11% response r a t e ) .  of v e n t r i c u l a r  t o c a i n i d e and q u i n i d i n e ,  r e d u c t i o n i n v e n t r i c u l a r e c t o p i c beats quinidine  Sonnhag,  to  al.,  of  the  1976).  resulting Both  gave w e l l - r e s o l v e d symmetrical peaks  and 5.9 minutes r e s p e c t i v e l y ,  and  both plasma and u r i n e were l i n e a r over  the  range  -  yg/mL.  17  of 0.25  to 20.0  urine.  The method was s u i t a b l e f o r measuring drug l e v e l s  However, no s p e c i f i c sensitivity  of  the h e p t a f l u o r o b u t y r y l  pharmacokinetic  tocainide.  s t u d i e s has  i n 50-100 uL of r a t p e r i o d of 3 h .  of  chromatographic  stability,  by these  sensitive  a flame  tocainide  serially  over a  extraction  the d e r i v a t i v e  formed,  (1980b) employed a  (1974) which c o n s i s t e d  derivative  (formed  of  from t o c a i n i d e  and  on 3% OV-17 or 10% OV-17 packed column  i o n i s a t i o n detector of  or e l e c t r o n c a p t u r e  detector.  t o c a i n i d e was s t a b l e only  for  1980b).  chromatographic method based upon f o r m a t i o n of a S c h i f f  base w i t h m e t h y l i s o b u t y l k e t o n e  tion,  of  to  authors.  the h e p t a f l u o r o b u t y r y l  24 h ( E l v i n e t a l . ,  extracted  t h i s method  by a n a l y s i s of  collected  structure  The m o n o h e p t a f l u o r o b u t y r y l d e r i v a t i v e  has  was r e p o r t e d l y  The a p p l i c a t i o n of  (1976) and E l v i n et a l . ,  heptafluoro-butyrylimidazole)  detector  A GLC method  derivative  m o d i f i e d GLC method of C o l t a r t e t a l . ,  A gas  appearance or  detector  been demonstrated  as w e l l as  or  up to 48 h .  T o c a i n i d e r e c o v e r y from the plasma by the  Lalka et a l . ,  using either  i n plasma  u s i n g an e l e c t r o n capture  plasma (dose 20 mg/kg)  have a l s o been s t u d i e d  a n a l y s i s of  peaks were observed  the method were i n c l u d e d .  and A x e l s o n , 1978)  down to 30 picograms  method used,  data r e g a r d i n g  and p r e c i s i o n of  (Venkataramanan analysis  No i n t e r f e r i n g  -  been r e p o r t e d  from a l k a l i n e  the r e s i d u e  i n a waterbath  and employing a n i t r o g e n  by Johansson et  al.,  (1982).  selective The amine was  samples w i t h dichloromethane and, a f t e r  was r e c o n s t i t u t e d  a t 8 5 ° C f o r 10 mins.  i n methylisobutylketone After  c o o l i n g to room  and  evaporaheated  temperature,  -  the s o l u t i o n was i n j e c t e d carbowax 20 M (10%)  d i r e c t l y i n t o the gas  packed column.  analog of t o c a i n i d e ,  chromatograph u s i n g a  The i n t e r n a l standard  The i n t e r n a l standard  base w i t h good chromatographic p r o p e r t i e s .  the t o c a i n i d e standard, ng/mL of  Schiff  used was an  c o n t a i n i n g 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 . Schiff  18 -  base was  13 m i n u t e s .  10 minutes and that  The l i m i t  a l s o formed a  The r e t e n t i o n time of of the i n t e r n a l  of q u a n t i t a t i o n f o r  t h i s method was 96  plasma.  High performance l i q u i d  chromatography has a l s o been employed f o r  the d e t e r m i n a t i o n of  tocainide  Meffin  Lagerstrom and P e r s s o n ,  et  al.,  1977;  i n the plasma  ( E l v i n et 1978).  al.,  Meffin  1980b; et  al.,  (1977) developed a very s e n s i t i v e HPLC method f o r measurement  of  tocainide  a  i n blood and plasma which i n v o l v e d the f o r m a t i o n of  fluorescent  d e r i v a t i v e with dansyl chloride  naphthalenesulphonyl chloride) 1:1  and an i s o c r a t i c  analysis  (mobile phase =  mixture of hexane and 2% methanol i n dichloromethane) u s i n g a  u-Bondapack-amino column and f l u o r e s c e n t shown to be u s e f u l levels of  (5-(dimethylamino)-l-  less  of  for  the drug (0.1  than 2%.  therapeutic  the measurement to 5.0  detection.  The method has  of t h e r a p e u t i c  or  been  subtherapeutic  ug/mL plasma) with a standard  deviation  Although the s e n s i t i v i t y was adequate to measure  plasma l e v e l s ,  the e x t r a c t i o n  procedure was t e d i o u s  and time  consuming. Lagerstrom and Persson  (1978) d e s c r i b e d  g r a p h i c t e c h n i q u e ( p e r c h l o r i c a c i d , methanol, 0.5:10:89.5)  an i o n - p a i r chromato-  dichloroethane,  u s i n g a P a r t i s i l column and UV d e t e c t i o n .  At a mobile  -  phase flow r a t e of about 6 minutes.  1.0  mL/min,  19 -  the r e t e n t i o n time of  Another HPLC method ( E l v i n e t  al.,  tocainide  1980b) employed an  octadecylsilane  (ODS) r e v e r s e - p h a s e column and UV d e t e c t i o n .  phase c o n s i s t e d  of 25% a c e t o n i t r i l e  r e c e n t HPLC method (Ronfeld et a l . ,  i n 0.05  The mobile  M NaClOit at pH 4 .  1982), a l s o an i o n - p a i r  0.01  M pentanesulfonic  a c i d , 0.01  T h i s method was used  tocainide,  for  the l a c t o x y l i d i d e  15:15:70 with the pH a d j u s t e d  the measurement  of  formed by o x i d a t i v e  t o c a i n i d e carbamoyl g l u c u r o n i d e which was f i r s t hydantoin.  Sedman e t a l . ,  standard,  converted  to a  The drug and c h e m i c a l l y s i m i l a r  under s a l t i n g - o u t  f o l l o w e d by internal  were e x t r a c t e d  was  treated  w i t h borate buffer  resulting derivatives  phase.  The authors  be c a r r i e d  the d e r i v a t i v e s product of  buffer  a l s o recommended that  out immediately before  the f l u o r e s c a m i n e  (pH 8.2)  were chromatographed  column u s i n g a methanol-phosphate  the r e a c t i o n of  for  fluorescamine  The  on an ODS r e v e r s e - p h a s e  (pH 7.0)  mixture as  the d e r i v a t i z a t i o n  HPLC a n a l y s i s .  several  of  The o r g a n i c  and f l u o r e s c a m i n e .  At room  i n t e n s i t y decreases s i g n i f i c a n t l y  can be s t o r e d  from plasma  c o n d i t i o n s obtained by s a t u r a t i o n  the aqueous medium w i t h sodium c h l o r i d e - s o d i u m c a r b o n a t e . extract  of  d e a m i n a t i o n , and  t o c a i n i d e w i t h fluorescamine  2-amino-6'-chloro-o-acetotoluidide,  with a c e t o n i t r i l e  two m e t a b o l i t e s  to  (1982) developed an HPLC method based on  pre-column d e r i v a t i z a t i o n of fluorometric detection.  consisting  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 , 3.5.  The more  technique,  used a r e v e r s e - p h a s e column, UV d e t e c t i o n and a mobile phase of  was  hours i f  i n 1-2  mobile reaction  temperature, h.  However,  cooled i n i c e .  w i t h racemic  tocainide  The  contains  - 20 -  two  asymmetric c e n t e r s  formed.  and t h e r e f o r e ,  two d i a s t e r e o m e r i c  There was no evidence of a s e p a r a t i o n  C.  S t e r e o s e l e c t i v e Drug A n a l y s i s  1.  Resolution  of  activity  of Diastereomers  in a stereoselective of enantiomers  may d i f f e r .  enantiomers are  d i s p o s i t i o n s t u d i e s are only i n t h e i r c h i r a l other c h i r a l  chromatographic  As a r e s u l t ,  Methods for  t h e r e f o r e necessary  to be c a r r i e d o u t .  properties  compounds)  manner.  (optical  and  are  GLC,  thus,  TLC).  reagents  the p h a r m a c o l o g i c a l  These have d i f f e r e n t  distribution,  the a n a l y s i s  Reactions leads  of  c h e m i c a l and p h y s i c a l  differentiate  a s s o c i a t e d with c h i r a l  i n c l u d e the requirement of an a c t i v e  pairs  differ  enantiomers  to formation of  A p p r o p r i a t e l y chosen d e r i v a t i z i n g reagents can  of d i f f e r e n t  the  by the normal  properties  i n p r i n c i p l e , s e p a r a b l e by chromatographic means  Disadvantages  of  stereoselective  S i n c e enantiomers  techniques of drug a n a l y s i s .  f o r m a t i o n of d i a s t e r e o i s o m e r i c rates  if  they are not r e s o l v a b l e  the chromatographic process as w e l l as enantiomers.  living  r o t a t i o n , i n t e r a c t i o n with  w i t h an o p t i c a l l y pure d e r i v a t i z i n g reagent diastereomers.  of  and t h e i r d i s p o s i t i o n ( a b s o r p t i o n ,  metabolism and e x c r e t i o n ) individual  are  diastereomers.  Drug enantiomers i n t e r a c t w i t h c h i r a l molecules organisms  derivatives  derivatives,  (HPLC,  facilitate  between  derivatization  f u n c t i o n a l group for  differences  i n the r e a c t i o n  of e n a n t i o m e r i c compounds and d i f f i c u l t i e s  o b t a i n i n g o p t i c a l l y pure reagents (Konig et a l . ,  1977).  the  in  In a d d i t i o n ,  -  the method cannot be employed i f c h e m i c a l l y and s t e r e o c h e m i c a l l y conditions.  21  -  the d i a s t e r e o m e r l c  not  s t a b l e under chromatographic  Although c a p i l l a r y columns were f i r s t  graphic analysis  mixture i s  (Raban and M i s l o w ,  used i n gas  1967), newer o p t i c a l l y  chromato-  active  reagents permit the use of packed columns. The most commonly used reagent S-prolyl 1966;  c h l o r i d e (TPC) ( W e l l s , 1970;  Dabrowiak and Cook, 1971).  S(-)-N-pentafluorobenzoyl al.,  1974),  ((S)-MTPA)  been N - t r i f l u o r o a c e t y l -  Westley e t a l . ,  Other reagents  (Gal et a l . ,  Gordis,  include  p r o l y l - l - i m i d a z o l i d i d e (PFBP) ( C h a t t e r j i e  et  1982;  G a l , 1977;  N i c h o l s , et a l . ,  1973).  These  a c y l a t i n g agents and d i s c r i m i n a t e between e n a n t i o m e r i c  amines and a l c o h o l s .  G a l (1977) used  amphetamine and e i g h t r e l a t e d  the l a t t e r  reagent  to  resolve  compounds.  D i r e c t R e s o l u t i o n o f Enantiomers With the r e c e n t i n t r o d u c t i o n of c h i r a l  d i r e c t GLC r e s o l u t i o n of enantiomers 1967)  first ester  coated  capillaries.  therefore analysed.  is  for  the s e p a r a t i o n  possible.  phases,  G i l - A v et a l . ,  the (1966,  of N-TFA ot-amino a c i d e s t e r s on w a l l -  The o p e r a t i n g  temperature  only h i g h l y v o l a t i l e d e r i v a t i v e s R e c o g n i z i n g the e s s e n t i a l  of amides which might i n c r e a s e  of  this  phase i s 9 0 ° C and  of amino a c i d s  r o l e of  these type of peptide e s t e r phases,  features  stationary  i n t r o d u c e d 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  lauryl  in  1968;  and S(-) o t - m e t h o x y - o t - t r i f l u o r o m e t h y l p h e n y l a c e t y l c h l o r i d e  reagents are  2.  has  can be  the -NHCO*CH(R)NH.CO-group  the search  for c e r t a i n  s e l e c t i v i t y as w e l l  as  structural thermal  -  stability  22  -  l e d 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 ( F e i b u s h ,  1971).  Subsequent  exhibited greater e f f i c i e n c y , r e t e n t i o n times stability  (Beitler  diamide phases d e r i v e d from L - v a l i n e  higher r e s o l u t i o n factors  and F e i b u s h ,  1976).  and reduced  To o b t a i n b e t t e r  thermal  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 c a r b o x y l group of the co-polymer of d i m e t h y l s i l o x a n e and carboxyalkylmethylsiloxane coated  on c a p i l l a r i e s  (Frank et a l . ,  and i s  designated  used  to separate e n a n t i o m e r i c drugs  Liu,  et a l . ,  1982), amino a c i d s  amino a l c o h o l s al.,  1980).  made i t  Chirasil-Val®  for  the f i r s t  al.,  incorporated  1978).  enantiomers  been 1978;  of h i g h and low v o l a t i l i t y and some 1977,  Frank et a l . ,  i n t o a w e l l known GLC s t a t i o n a r y  Frank et  Chirasil-Val®  spectrometer  of enantiomers  L - v a l i n e - t e r t . b u t y l a m i d e has  1979;  of  time to employ a mass  f o r the a n a l y s i s  phenylmethylsilicone (0V-225).  and has  (Frank et a l . ,  In a d d i t i o n , the h i g h thermal s t a b i l i t y  coupled to GLC system  3.  T h i s phase was w a l l  and m e t a b o l i t e s  (Solomon and W r i g h t ,  possible  F r a n k , et  as  1977).  ( L i u et  al.,  1981;  a l s o been  phase, p o l y c y a n o p r o p y l  The r e s o l u t i o n of n o r e p h e d r i n e  was accomplished on t h i s  phase (Saeed et  al.,  1979).  R e s o l u t i o n o f Enantiomers by HPLC L i k e GLC methods, HPLC has a l s o been used  enantiomers. separation (Baczuk,  et  to  resolve  Most of the e a r l i e r methods r e p o r t e d have d e a l t  of amino a c i d s al.,  1971;  u s i n g a v a r i e t y of d e r i v a t i z i n g  Lefebvre et  chromatography has been used  al.,  to r e s o l v e  1978).  with  reagents  More r e c e n t l y ,  e n a n t i o m e r i c amines  ion-pair  (Pettersson  -  and S c h i l l ,  1981).  T h i s method i s  a c h i r a l c o u n t e r - i o n added sulphonic  a c i d as  enantiomers  (Hermansson  as  and Bahr,  1980).  the s t a t i o n a r y  of w a r f a r i n  phase.  detection  approach has been used  i n the plasma ( B a n f i e l d  The method permits  of w a r f a r i n  stationary  enantiomers  stationary  by HPLC.  phases such as  aminopropyl-silica gel enantiomeric  enantiomers  to  and Rowland, esters,  characterization  of  silica the  following administration  Stereospecific Since  phases a l s o have been developed  for  P i r k l e et  chiral  al.,  (1980) developed  3,5-dinitrobenzoyl  of  the  resolution  p h e n y l g l y c i n e attached  f o r l i q u i d chromatographic  amines and a l c o h o l s .  available in either  T h i s column i s  to  s e p a r a t i o n of now c o m m e r c i a l l y  the i o n i c a l l y or c o v a l e n t l y bonded  form.  Radioimmunoassays  antibodies  they b i n d to enantiomers is  followed by  drug. Chiral  it  A similar  was  diastereomeric  c a r b o b e n z y l o x y - L - p r o l i n e , w i t h subsequent s e p a r a t i o n u s i n g  racemic  4.  by f o r m a t i o n of a  resolve  Propranolol  T h i s method i n v o l v e d the f o r m a t i o n of d i a s t e r i o m e r i c  pharmacokinetics  of  these authors were a b l e to and p r o p r a n o l o l .  with  camphor  on a r e v e r s e - p h a s e column w i t h f l u o r o m e t r i c  r e s o l v e enantiomers  using  enantiomers  chromatography  Using (+)  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  chromatography  1983).  to the mobile phase.  the c o u n t e r - i o n ,  into i t s  -  based on i o n - p a i r  of a l p r e n o l o l , m e t o p r o l o l  also resolved derivative  23  are c h i r a l compounds, exhibit  differing  the complexes  stability  formed when  constants.  p o s s i b l e to prepare a n t i s e r a to i n d i v i d u a l enantiomers  Thus,  of a drug  -  and  use  the a n t i s e r a  selectivity other.  24 -  f o r enantiomer a n a l y s i s .  In g e n e r a l ,  excellent  can be achieved f o r one enantiomer i n the presence  R e c e n t l y , Midha et a l . ,  radioimmunoassay  (1983) developed a  f o r d- and 1-ephedrine  of  stereospecific  i n human plasma and have used  the technique to f o l l o w the plasma concentration.-:time curve a f t e r oral  5.  dose of  the  R e s o l u t i o n o f T o c a i n i d e Enantiomers  chiral  chromatographic method based on c o n v e r s i o n of  amine to a diastereomer  (1982).  gas  reagent used was  c h l o r i d e (MTPA).  reagent and heated at  formed.  (1.0  has been r e p o r t e d by G a l , et  The c h i r a l d e r i v a t i s i n g  trifluorophenylacetyl  The  60 - 6 5 ° C  in this  lower l i m i t  of s e n s i t i v i t y  enantiomer and t h e r e f o r e mL u r i n e )  had to be used.  the d i a s t e r e o m e r  acylation  was  reaction.  by an acid-wash w i t h h y d r o c h l o r i c a c i d  N) f o l l o w e d by washing w i t h sodium carbonate chromatographic a n a l y s i s  al.,  (S)-ct-methoxy-ct-  f o r 30 minutes  P y r i d i n e was used as a c a t a l y s t  d e r i v a t i v e had to be i s o l a t e d  the  When t o c a i n i d e was mixed w i t h  of the d e r i v a t i v e s ,  column (3% OV-17) and a n i t r o g e n - p h o s p h o r u s  S(+)  an  racemate.  A gas-liquid  this  the  s o l u t i o n (15%).  a c o n v e n t i o n a l packed  detector  of the assay was 1.0  was u s e d .  ug/mL f o r  a r e l a t i v e l y l a r g e sample s i z e The r e t e n t i o n times  t o c a i n i d e was 13 mins and that  of R(-)  of  The  each  (1 mL serum or 1  the MTPA d e r i v a t i v e of  tocainide,  11.3  mins.  D i r e c t R e s o l u t i o n of T o c a i n i d e Enantiomers R e c e n t l y McErlane and P i l l a i  (1983) developed a  chromatographic method f o r the d i r e c t  For  r e s o l u t i o n of  gas-liquid  tocainide  -  enantiomers  employing a f u s e d - s i l i c a  optically active butylamide.  stationary  al., of  for  1980).  as  as  saliva  racemate. (electron  and amino a l c o h o l s  excellent  r e s o l u t i o n of the  their heptafluorobutyrate  measurement  et a l .  derivatives.  (1984) used t h i s  of t o c a i n i d e enantiomers  f o l l o w i n g o r a l or i n t r a v e n o u s The lower l i m i t capture  stationary  phase  are  C h i r a s i l - V a l ® and have been demonstrated  r e s o l u t i o n of amino a c i d s  (1983) and P i l l a i  simultaneous and  c a p i l l a r y column coated with an  phase, N - i s o b u t y r y l - L - v a l i n e - t e r t . -  T h i s phase o f f e r s  tocainide  Pillai  -  C a p i l l a r y columns coated with t h i s  commercially a v a i l a b l e effective  25  detector)  of  et  enantiomers  McErlane and  technique f o r  the  i n human plasma,  a d m i n i s t r a t i o n of  of s e n s i t i v i t y  (Frank,  to be  urine  the  the assay was 25 ng/mL  f o r each enantiomer u s i n g the s p l i t - m o d e  of  injection.  D.  Renal F a i l u r e and Drug A c c u m u l a t i o n In view of  the extent  tocainide  (40% of  drug i n normal s u b j e c t s and (20-70%) i n d i s e a s e  states)  dose as  intact  and  major m e t a b o l i t e  its  of u r i n a r y e x c r e t i o n of  TOCG (25-40% d o s e ) ,  d y s f u n c t i o n on the e l i m i n a t i o n of  the e f f e c t  of  renal  these substances should be  to permit r a t i o n a l dosing adjustment  quantitated  i n r e n a l d i s e a s e and d u r i n g  dialysis. The  time p e r i o d d u r i n g which a drug s t a y s i n the body and a l s o  the i n t e n s i t y and d u r a t i o n of p h a r m a c o l o g i c a l the r a t e and extent elimination. their  of drug a b s o r p t i o n ,  Some drugs  are  cleared  unchanged form w h i l e others  effects  are  functions  d i s t r i b u t i o n , metabolism and  from the body predominantly i n  are p a r t i a l l y or c o m p l e t e l y  of  -  metabolised kidneys. form,  and then c l e a r e d  partially function  metabolised,  a f u n c t i o n of i n t e g r i t y is  compromised  converted lead renal  to t o x i c  failure  a variety related  levels  to a m e t a b o l i t e ,  to t o x i c e f f e c t s . is  its  from the body i n unchanged  overall  pharmacologic  of r e n a l f u n c t i o n .  tend to be r e t a i n e d  w i t h repeated d o s i n g .  factors.  The i n a b i l i t y of  without i n c r e a s e d  blood l e v e l s and i t s  (2-4  fold)  1980)  and r a t  (Tam et  insufficiency  (glomerular  ampicillin is  increased  35-67 h (McHenry et  al.,  (Drayer et in total  al.,  al.,  chronic require  al.,  from 1 h to al.,  1977).  body c l e a r a n c e  intact  to obtain  Renal (with  i n man (Bateman  drug.  failure  that  and both  In r e n a l  r a t e < 5 mL/min) the h a l f - l i f e 10-18  h (Kunin,  1970);  1959); g e n t a m i c i n  1972).  of  tetracycline  from 2.3  to  1971); vancomycin from 6 to 200 h ( K u n i n , and chlorpropamide  for  have been shown  1981), d e s p i t e the f a c t  filtration  Lindholm and Murray, 1966) et  may a l s o  toxicity.  N-acetyl metabolite  the dose as  to 57-108 h (Kunin et  (Petitpierre  dosage adjustment to  i n c r e a s e i n t\/2) of metoclopramide  s p e c i e s o n l y e x c r e t e 20% of  from 8.5  active  i n renal dysfunction  caused l a r g e r e d u c t i o n s  is  of  these p a t i e n t s  adequate t h e r a p e u t i c  Gokhal,  a drug  t h e i r b a s i c r e n a l c o n d i t i o n and  careful  concomitant  If  the m e t a b o l i t e  e x c r e t e drugs adequately r e q u i r e s  to accumulate  i n the body and  concern i n o l d e r p a t i e n t s who may  both f o r  is  eliminated  The prolonged and p r o g r e s s i v e nature  of m e d i c a t i o n s ,  Procainamide  profile  When r e n a l  drugs  then accumulation of  of p a r t i c u l a r  or u n r e l a t e d  cleared  through r e n a l d i s e a s e ,  predominantly by the kidneys w i l l may accumulate  -  from the body predominantly by the  Thus, whether a drug i s  or e x t e n s i v e l y  26  from 36 to 200 h  1967;  -  In a d d i t i o n to the parent pharmacologically renal of  excretion  active,  is  27  drug,  -  drug m e t a b o l i t e s which may be  can a l s o accumulate  a major  i n renal  pathway of e l i m i n a t i o n .  be p a r t l y  metabolites.  explained  Norpethidine i s  a n a l g e s i c but more convulsant metabolite cancer  activity  may,  irritability  of p e t h i d i n e w i t h l e s s  (Denean and N a k a i ,  but r a p i d l y i n p a t i e n t s w i t h r e n a l  1961).  norpethidine  (Szeto et a l . ,  1976).  i n most Severe  failure  a s s o c i a t e d w i t h e x c e s s i v e accumulation of chlorophenoxyisobutyric f r e e and a c e t y l a t e d  acid (Pierides  sulphonamides  receiving  the f r e e a c i d  et a l . ,  1975).  side effects  clofibrate  Accumulation of failure  i n renal  insufficiency,  d i s t r i b u t i o n of drugs such as  of drugs  physiological  and  the pharmacokinetic  parameters of  drugs,  The volume of  d i g o x i n (Reuning et a l . ,  1973),  m e t h i c i l l i n and l i n c o m y c i n ( G i b a l d i and P e r r i e r , to decrease due to changes i n t h e i r i n renal  and/or  certain  making dosage m o d i f i c a t i o n even more d i f f i c u l t .  characteristics  is  1975).  changes a l s o a l t e r  been r e p o r t e d  are  (severe nausea and  In a d d i t i o n to the r i s k of t o x i c • a c c u m u l a t i o n their metabolites  of  metabolite,  i n patients with renal  a s s o c i a t e d w i t h an i n c r e a s e i n t o x i c Weinstein,  associated with high l e v e l s  The severe muscle weakness and  tenderness seen i n p a t i e n t s w i t h r e n a l  cephalexin,  failure.  This  and t w i t c h i n g has been noted i n uremic p a t i e n t s who have  been t r e a t e d w i t h p e t h i d i n e and these are  anatomical  for  drug  accumulates slowly w i t h m u l t i p l e doses of p e t h i d i n e  patients,  vomiting,  failure  by accumulation of a c t i v e  a metabolite  if  The h i g h i n c i d e n c e  adverse drug r e a c t i o n s seen i n p a t i e n t s w i t h r e n a l  some d r u g s ,  insufficiency  impairment.  1972)  distribution  have  -  28  -  R e d u c t i o n i n r e n a l blood flow and glomerular associated with retention  of  sodium and w a t e r .  an i n c r e a s e i n the volume of d i s t r i b u t i o n i n steady s t a t e plasma c o n c e n t r a t i o n .  function  of  this  than i n normal s u b j e c t s .  For i n s t a n c e , L e t t e r !  p h e n y t o i n and w a r f a r i n  higher  and anatomical  given o r a l l y . 1972)  patients  the plasma  and d o x y c y c l i n e  reviewed  than normal s u b j e c t s a f t e r  1978).  Recent  1971;  The f i r s t  (1971) who observed failure  Odar-Cederlof  al.,  1967)  receiving  Reidenberg,  1974;  r e t i c u l u m of  observation  of  this  and  renal  Borga  than i n normal s u b j e c t s . (1974) who proved  was  tract of  caused  drugs et  lower i n r e n a l  similar  oral  Reidenberg,  s o r t was  more r a p i d e l i m i n a t i o n of  by the l i v e r  plasma  (Petitpierre  doses. has et  that drug  the l i v e r are  been al., oxidations  accelerated  that of L e t t e r i  et  in al.  phenytoin i n p a t i e n t s w i t h  T h i s was confirmed by  that the r a p i d e l i m i n a t i o n of  i n uremic p a t i e n t s was due to r a p i d m e t a b o l i c oxidised  are  s t u d i e s have l e d to the o b s e r v a t i o n  o c c u r r i n g i n the endoplasmic uremia.  of chloropropamide  of kidney d i s e a s e on drug metabolism  (Reidenberg,  al.,  disease.  to some degree the a b s o r p t i o n  (Fabre et  to  concentrations  changes to the g a s t r o i n t e s t i n a l  Serum c o n c e n t r a t i o n s  The i n f l u e n c e  et  were lower and the volume of d i s t r i b u t i o n  by c h r o n i c uremia may a f f e c t  renal  that  i n uremic p a t i e n t s than i n p a t i e n t s without r e n a l  Functional  al.,  of p h e n y t o i n ,  S i m i l a r l y Odar-Cederlof  (1977) r e p o r t e d  are  drugs and decrease  drug were lower i n p a t i e n t s w i t h impaired  (1974) and O d a r - C e d e r l o f of  rate  These events can l e a d  of c e r t a i n  (1971) found that f o l l o w i n g bolus i n j e c t i o n s concentrations  filtration  clearance.  phenytoin  Another drug  that has been found to have a c c e l e r a t e d  metabolism  -  in  patients with renal  e l i m i n a t i o n r a t e has  failure  An i n c r e a s e d  i n both d i a l y s e d  failure  and  ( L i c h t e r et a l . ,  antipyrine  undialysed  1973;  Maddocks  et  1975).  E.  Glucuronldation i n Renal F a i l u r e G l u c u r o n i d e s are  ionised  cant  acidic  i n plasma and u r i n e ,  the k i d n e y s .  compounds  extent i n t o of  and are r e a d i l y  (Levine,  1978;  the b i l e ,  R o l l i n s and K l a s s e n ,  into  glucuronide  the b i l e .  glucuronides  excreted  may become s u b s t r a t e s  the g a s t r o i n t e s t i n a l parent  which i s  tract,  Couchie,  can a l s o be c a r r i e d  extensively  Wakabayashi,  investigated  however,  diminished  Hartiala,  out by lysozomal  particularly  have  (phenolic  1970).  acid,  and  excretion of  the  excretion,  glucuronidase of  the  of  liberated  1973).  Hydrolysis  ^-glucuronidase spleen,  organs (Levy and  Glucuronide conjugation  i n p a t i e n t s with r e n a l  shown that  if  l i v e r , kidney,  e p i t h e l i u m and endocrine and r e p r o d u c t i v e  1966;  true  the m i c r o b i a l  1971;  signifi-  e l i m i n a t i o n of  Following b i l i a r y  permitting reabsorption  p r e s e n t i n most t i s s u e s ,  intestinal  biliary  Although excretion  not n e c e s s a r i l y  for  compound ( O k o l i c s a n y e e t a l . ,  glucuronides  rats,  into  is  to a  chemical s t r u c t u r e  the u r i n e r e s u l t s i n the i r r e v e r s i b l e this  fully  from the body by  a l s o excreted  the e x t e n t of  1979).  the conjugate from the body, is  eliminated  w i t h m o l e c u l a r weight,  the g l u c u r o n i d e d e t e r m i n i n g  glucuronides  (pka 3.5-4) which are  G l u c u r o n i d e s of some drugs are  polarity  of  -  antipyrine.  been observed  patients with chronic renal al.,  is  29  failure.  has  not  Studies  been  in  the g l u c u r o n i d a t i o n process may be  p-aminobenzoic  acid),  increased  (naphthol)  -  or unaffected  (4-methylumbelliferone)  (Canalese et a l . , Despite failure,  1980;  i n experimental r e n a l  Howie and Bourke, 1979;  the s c a r c i t y  by t h i s  hydrolysis  failure  of the c o n j u g a t e ,  compound ( V e r b e e c k , biotransformed 1979b).  1982).  Despite  this,  the t o t a l body c l e a r a n c e  et a l . ,  include altered  1979;  However, r e c e n t to  active  clofibrate  is  patients  form of c l o f i b r a t e ,  (Faed et a l . ,  single-dose study,  decreased  Verbeeck et  i n patients al.,  al.,  tract  and r e a b s o r p t i o n  1979;  Gugler,  salicylate  with renal  failure  1979). D i f l u n i s a l a l s o  1975).  Possible  patients  (Verbeeck et a l . ,  has  biliary  f o l l o w e d by h y d r o l y s i s i n  the g l u c u r o n i d e s ,  as  and has  1980;  been Levy,  Glucuronide accumulation i n renal  a l s o been shown f o r  1976)  forms  mechanisms  the e s t e r - t y p e of g l u c u r o n i d e of CPIB ( F a e d , 1977).  was  i t was shown that  of unchanged d i f l u n i s a l ,  of one or both of  et  of  g l u c u r o n i d a t i o n of d i f l u n i s a l i n r e n a l f a i l u r e ,  De Schepper et a l . ,  failure  as  of d i f l u n i s a l , a r e c e n t l y developed  1977;  deconjugation for  1978).  ( C P I B ) , the a c t i v e  e x c r e t i o n of one or both of the g l u c u r o n i d e s  described  categorized  to e x c r e t e a g l u c u r o n i d e may l e a d  an e s t e r - t y p e g l u c u r o n i d e (Tocco et  systemic  with r e n a l  the plasma e l i m i n a t i o n h a l f - l i f e  was s i g n i f i c a n t l y  the i n t e s t i n a l  1972).  to an e s t e r - t y p e g l u c u r o n i d e (60% of dose) (Gugler  S i m i l a r l y , i n a recent,  (De Schepper  al.,  l e a d i n g to accumulation of the  f o l d longer i n renal f a i l u r e  analgesic,  generally  The a n t i l i p e m i c agent,  chlorophenoxyisobutyric acid  1979a).  Leber et  failure  i n experimental data i n patients  d i s e a s e s t a t e (Reidenberg,  s t u d i e s have shown that  2-6  -  the g l u c u r o n i d a t i o n process i s  unaffected  al.,  30  and oxazepam  the b e n z o d i a z e p i n e s ,  ( O d a r - C e d e r l o f et  al.,  lorazepam 1977).  -  F.  E n a n t i o s e l e c t l v i t y i n Drug O p t i c a l isomers  31  Disposition  of many drugs  pharmacological a c t i v i t i e s  -  have been shown to d i f f e r  due to t h e i r a b i l i t y to r e a c t  selectively  w i t h an asymmetric c e n t e r i n the b i o l o g i c a l system (Casy, al.,  1975;  Ramasastry,  1973).  The observed d i f f e r e n c e s  a c t i v i t y may be due to a d i f f e r e n c e drug-receptor isomers  combination.  are s e l e c t e d  system before  i n the p r o p e r t i e s  Differences  Patil  et  in biological  of  the optical  by some o t h e r asymmetric c e n t e r i n the b i o l o g i c a l  the isomer reaches  the s p e c i f i c  receptor.  selective  during transport  Ames et  1978).  1970;  i n d i s t r i b u t i o n occur as  to o p t i c a l l y a c t i v e p r o c e s s e s such as  selective  in their  ( P a t i l et a l . ,  metabolism (Jenner  An o p t i c a l l y a c t i v e  1975;  and T e s t a ,  1980;  T h i s may be due  p e n e t r a t i o n of membranes  al.,  1977;  G r a y , 1976),  or  Low and C a s t a g n o l i ,  drug may not be s u b j e c t e d  to a l l of  these  p r o c e s s e s but they may c o n t r i b u t e 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 f e c t  of  one i s o m e r .  1.  Enantioselective The  Distribution  two main f a c t o r s  i n f l u e n c i n g the plasma h a l f - l i v e s  phenprocoumon and w a r f a r i n enantiomers proteins  and t h e i r b i o t r a n s f o r m a t i o n  Schmidt and Jahnchen, by  various  selectively  1977)  t h e i r b i n d i n g to serum  (Breckenridge and Orme,  Jahnchen et a l . ,  organs may be e n a n t i o s e l e c t i v e  (Schmidt and Jahnchen,  uptake i s  1977;  are  as  1976).  1972;  The uptake of  drugs  shown by phenprocoumon.  where the more potent enantiomer  taken up by the l i v e r .  of  Another example of  is  stereoselective  seen w i t h the a n t i m u s c a r i n i c drug b e n z e t i n i d e ,  the  -  p h a r m a c o l o g i c a l l y potent  S(+)  up i n t o guinea p i g c a r d i a c to be almost  enantiomer of which i s  tissues.  much s m a l l e r  therefore extent  al.,  1976).  Another example  Following intravenous  were m o n i t o r e d .  form was three  times  form.  found i n the heart was the (-) selectively  isomer remains  cardiac uptake. isomer.  It  accumulated i n i t s  i n the blood and i s  i n the heart  and b r a i n of mice (Levy et a l . ,  documented f o r propoxyphene.  infused  form  is  the p r o p r a n o l o l the  active the  (Kawashima  1976).  Only the (+)  this  perfused  An i n t e r e s t i n g  and e n a n t i o m e r i c i n t e r a c t i o n has  synergistic  the two enantiomers.  i n the i s o l a t e d  from the  organ of a c t i o n whereas  isomer shows  A 20 mg/kg dose of t h i s  The e x p l a n a t i o n f o r  d i s p o s i t i o n of  much f a s t e r  Most of  et  rat  effect  case  been  analgesic  isomer i n the r a t  e q u i - a c t i v e w i t h a combination of two enantiomers each.  antipode.  uptake of p r o p r a n o l o l has a l s o been found  s t e r e o s e l e c t i v e l i v e r uptake  i n the r a t .  the  the serum l e v e l  r a p i d l y metabolised  Stereoselective  activity  of i t s  of  thus appears that  a l . , 1976).  of  a d m i n i s t r a t i o n of  5 minutes,  form disappeared  B-adrenergic  The i n i t i a l l y lower l e v e l of the (-)  e x p l a i n e d by a h i g h l y s e l e c t i v e  (+)  After  but to a  binding sites  concentrations  lower than that  D u r i n g the f o l l o w i n g 4 h , the (+)  enantiomer i s  The n o n - s p e c i f i c  p r o v i d e d by the  the serum and heart  i n d i v i d u a l enantiomers  serum than the (-)  is  taken  accumulation was found  than the p h a r m a c o l o g i c a l r e c e p t o r  the racemate to r a t s ,  the S(-)  selectively  display a certain stereoselectivity  b l o c k i n g agent, p r o p r a n o l o l .  of  The c a r d i a c  e n t i r e l y due to n o n - s p e c i f i c b i n d i n g .  binding s i t e s  (Gray et  32 -  at a dose of  is 10 mg/kg  was found i n the  When dextro-propoxyphene  was  l i v e r , over 98% of the drug was  -  extracted  in a single  p e r f u s a t e at  pass.  33  -  When the l e v o - i s o m e r was added to  the same c o n c e n t r a t i o n , the e x t r a c t i o n of  was reduced to l e s s than 90%. where the plasma l e v e l s upon simultaneous  A similar  situation  of dextro-propoxyphene  dosing of i t s  enantiomer.  the  It  dextro-isomer  prevails  increased  in vivo  several-fold  therefore  appears  the levo-propoxyphene must be p r e f e r e n t i a l l y bound to uptake metabolic  s i t e s w i t h i n the l i v e r , r e s u l t i n g  dextro-propoxyphene increased  2.  systemic  i n t o the systemic  In  that  or  i n the r e l e a s e of  c i r c u l a t i o n and hence  a v a i l a b i l i t y (Murphy et a l . ,  Enantioselective  the  its  1976).  Metabolism  most i n s t a n c e s ,  s t e r e o s e l e c t i v e drug d i s t r i b u t i o n may occur  c o n c o m i t a n t l y with s t e r e o s e l e c t i v e metabolism and the l a t t e r  often  determines  i n the  the o v e r a l l d i s p o s i t i o n of the d r u g .  d i s p o s i t i o n of  p r o p r a n o l o l enantiomers  (Kawashima et a l . , metabolism,  1976;  have a l s o been r e p o r t e d  George et a l . ,  1972;  s t e r e o s e l e c t i v e uptake have been noted f o r of  differences  have been r e p o r t e d  1975).  only f o r  1975).  Slower  formation and more  the S(-)  p r o p r a n o l o l have been c h a r a c t e r i z e d  isomer. but  Numerous  enantioselective  the O - g l u c u r o n i d e ( E h r s o n ,  E n a n t i o s e l e c t i v e metabolism of the a n t i a r r h y t h m i c agent,  d r o b u l i n e has been r e p o r t e d plasma l e v e l s  of the (-)  (Murphy et a l . ,  1977).  T h r e e - f o l d higher  isomer were observed when dogs were dosed  the racemate or w i t h i n d i v i d u a l the (+)  Ehrson,  l o n g e r h a l f - l i f e , more 0 - g l u c u r o n i d e  metabolites  Differences  enantiomers.  isomer appears to account  for  with  More r a p i d metabolism of  the d i f f e r e n c e s  i n plasma  levels  -  of  the two e n a n t i o m e r s .  demonstrated  Chiral  less  and  may be r e s p o n s i b l e  a c t i v e R(-)  for i t s  bupivacaine i s  S(+)  configurational  observed  and T e s t a  inversion is  stability  postulated  for  generally  the more t o x i c  1973).  c o n s i d e r e d as  Stereochemical  1975).  i n the mouse,  were shown to be  Following to  several  dextro-rotatory  rat  and G i l b e r t ,  There appears to be almost (-)  and guinea p i g (Adams et a l . ,  enantiomer to the (+)  process of c o n f i g u r a t i o n a l  1974; complete  form to the much more a c t i v e  c o m p o s i t i o n of the products  (-)  undergoes  i n v e r s i o n was  ibuprofen.  excreted  a d m i n i s t r a t i o n of i b u p r o f e n , r e v e a l s a s i g n i f i c a n t c o n v e r s i o n of  involving  i n d i v i d u a l isomers  isomer (Brooks  i n v e r s i o n of the much l e s s a c t i v e  stereochemical  metabolism have  The unchanged drug e x c r e t e d was a l s o found to be  e n r i c h e d i n the d e x t r o - r o t a r y V a n g i e s s e n and K a i s e r ,  R(-)  Process  the a n t i - i n f l a m m a t o r y d r u g ,  the major u r i n a r y m e t a b o l i t e s  enantiomer  for  of one isomer w h i l e the other isomer  a d m i n i s t r a t i o n of racemic i b u p r o f e n or i t s  et a l . ,  toxicity.  (1980).  slow or r a p i d c o n v e r s i o n to the former.  (Mills  p r e f e r e n t i a l l y hydrolyzed  Numerous other examples of e n a n t i o s e l e c t i v e  Configurational  1977).  enantiomer of both mepivacaine and  I n v e r s i o n o f C o n f i g u r a t i o n as a M e t a b o l i c  species,  (De Jong  g r e a t e r methemoglobinemic  more r a p i d than that  been r e p o r t e d by Jenner  first  agents have a l s o been  disposition  isomer of p r i l o c a i n e i s  A b s o r p t i o n of the a c t i v e  3.  l o c a l anesthetic  to e x h i b i t e n a n t i o s e l e c t i v e  The  antipodes.  34 -  i n v e r s i o n as  1976).  The  i n human u r i n e but  form ( K a i s e r  et  (+)  after  incomplete al.,  1976).  e x e m p l i f i e d by i b u p r o f e n  is  The  -  35 -  common to a number of a n t i - i n f l a m m a t o r y 2 - a r y l p r o p i o n i c C i c l o p r o f e n undergoes  (-)  man ( K r i p a l a n i et a l . , r e c e n t l y that R(-)  to (+)  1976;  the r a t s  c o n v e r s i o n i n the dog, r a t ,  Lan et a l . ,  benoxaprofen i s  enantiomer by r a t s and humans.  this  occurs w h i l e p a s s i n g the gut w a l l ,  1976).  It  is  monkey and  a l s o shown  s t e r e o c h e m i c a l ^ i n v e r t e d to the  1980).  These authors  t r a n s f o r m a t i o n does not occur i n the l i v e r through the gut w a l l s .  the extent  If  of i n v e r s i o n of R(-)  but  i n v e r s i o n does occur i n  benoxaprofen i n v a r i o u s  to the degree of e n t e r o h e p a t i c c i r c u l a t i o n .  Thus r a t s , which e x c r e t e  benoxaprofen p r i m a r i l y i n the  ( C h a t f i e l d and Green,  1978)  compound, are r a p i d i n v e r t e r s .  feces v i a  the  and have the p o t e n t i a l to reabsorb Whereas humans, who e x c r e t e  p r o p o r t i o n of the dose i n the u r i n e (Smith et a l . , c o r r e s p o n d i n g l y l e s s b i l i a r y e x c r e t i o n , are  in  also  s p e c i e s c o u l d be r e l a t e d  bile  S(+)  The r a t e of i n v e r s i o n i s much f a s t e r  than i n human (Simmonds et a l . ,  suggested that  acids.  1977)  the  a high  with  slow i n v e r t e r s of  the  compound.  G.  S p e c i f i c Alms o f t h e P r o j e c t 1. The chemical s t r u c t u r e  and t h e r e f o r e  of t o c a i n i d e c o n t a i n s an asymmetric  the molecule can e x i s t  i n two i s o m e r i c forms.  m i x t u r e , which c o n t a i n s equal p r o p o r t i o n s of l e v o - r o t a t o r y isomers, agent.  is  The racemic  the d e x t r o - and  used t h e r a p e u t i c a l l y as  an a n t i a r r h y t h m i c  Although the a n t i a r r h y t h m i c potency of t o c a i n i d e  has not been s t u d i e d i n man, the R(-)enantiomer i s potent  center  enantiomers  t h r e e - f o l d more  than the S(+)isomer as an a n t i a r r h y t h m i c agent i n a mouse model.  - 36 -  Differences  i n a n t i a r r h y t h m i c a c t i v i t y between the enantiomers  been demonstrated plasma l e v e l s  of  i n c o r o n a r y - l i g a t e d dogs. the enantiomers  observed i n mice and r a t s . employing d i a s t e r e o i s o m e r used i n these s t u d i e s .  as w e l l as  In a d d i t i o n , d i f f e r e n c e s  in  u r i n a r y e x c r e t i o n have been  A packed-column gas  chromatographic method  f o r m a t i o n and n i t r o g e n - s e l e c t i v e  The lower l i m i t  have a l s o  d e t e c t i o n was  of s e n s i t i v i t y of the assay  method was 1 mg/mL plasma f o r each enantiomer and t h e r e f o r e would not be suitable  f o r the c h a r a c t e r i s a t i o n  t o c a i n i d e enantiomers administered. stereoselective  of pharmacokinetic parameters  i n human, e s p e c i a l l y when low doses  T h i s has n e c e s s i t a t e d  of  individual  isomers  to measure  in biological fluids.  chromatographic methods are  superior  are  the development of a  assay method that not only would r e s o l v e  but a l s o would have h i g h s e n s i t i v i t y  the two  Since c a p i l l a r y column  to c o n v e n t i o n a l gas  detection,  the aim was to develop a c a p i l l a r y gas  for  2.  w i t h o p t i c a l l y pure  suitable  the need f o r  reagents.  Since t o c a i n i d e had never been assayed by a c a p i l l a r y column  chromatographic method u n t i l  necessary  capillary  d e t e c t i o n would be most  enantiomer d i s p o s i t i o n s t u d i e s s i n c e t h i s would o b v i a t e  f o r m a t i o n of diastereomers  gas  chromatographic  r e s o l u t i o n of enantiomers on c h i r a l  columns combined w i t h e l e c t r o n capture  gas  chromatographic  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  Moreover, d i r e c t  isomers  the low nanogram l e v e l s  procedures  method.  of  this  to study the f e a s i b i l i t y  extremely low l e v e l s the r e s u l t s  study was i n i t i a t e d , i t  was  of the technique by a n a l y z i n g  of the drug i n plasma and u r i n e and  obtained with published data.  It  comparing  was a l s o necessary  to  -  validate  -  the s t e r e o s e l e c t i v e assay by an independent d e t e r m i n a t i o n of  the racemate.  T h i s has n e c e s s i t a t e d  non-stereoselective decided  37  to use  developmental  the development of a  c a p i l l a r y column gas  small laboratory phase i n order  animals  Tocainide is  reported  hydantoin.  It  was  initial  pharmacokinetic  reports. by a n o v e l pathway  The assignment of  was based on i n d i r e c t evidence  3-(2,6-xylyl)-5-methyl  r a t s d u r i n g the  to be m e t a b o l i s e d  a g l u c u r o n i d e of N - c a r b o x y t o c a i n i d e . this metabolite  such as  to compare e a r l y  measurements with p r e v i o u s l y p u b l i s h e d 3.  chromatographic method.  such as  the s t r u c t u r e conversion  This hydantoin d e r i v a t i v e  to of  to  can be  formed not only from the g l u c u r o n i d e of N-carboxy t o c a i n i d e but a l s o by r e a c t i o n between phosgene and t o c a i n i d e (1982).  T h e r e f o r e i t was of i n t e r e s t  sufficiently proposed will in  large quantity  structure.  to gather  shown by Johansson et  to i s o l a t e  this metabolite  additional  evidence  A v a i l a b i l i t y of a pure sample of the  help i n developing a d i r e c t  the u r i n e .  as  Such methods  assay method f o r  can then be used  for  for  al., in  the  glucuronide  the i n t a c t  glucuronide  the study of  stereoselective glucuronidation. 4. lidocaine  While many a n t i a r r h y t h m i c drugs such as and m e x i l e t i n e have been measured  possibility  of s a l i v a r y  investigated.  was of  interest  e x c r e t i o n of t o c a i n i d e  into saliva  existed  and plasma l e v e l s .  between  saliva  i n the s a l i v a ,  e x c r e t i o n of t o c a i n i d e  Therefore i t  of a s t e r e o s e l e c t i v e assay f o r  procainamide, the  has never been  to study the extent  and to determine  if  In a d d i t i o n ,  of  a correlation the  the d e t e r m i n a t i o n of s a l i v a r y  application composition  -  of  enantiomers  salivary chiral  would r e v e a l  excretion.  there i s  -  any s t e r e o s e l e c t i v i t y  in  Such a p o s s i b i l i t y has never been i n v e s t i g a t e d  with  drugs. 5.  Tocainide is  the a d m i n i s t e r e d d o s e . in  if  38  the u r i n e as  excreted  i n the u r i n e to the extent  Approximately 30% of the dose i s  tocainide glucuronide.  i n patients  to study the extent  It  of accumulation of  was of  excreted  interest,  tocainide  w i t h r e n a l d y s f u n c t i o n and to determine i f  process removed the d r u g .  also  Therefore tocainide e l i m i n a t i o n  would depend on i n t e g r i t y of r e n a l f u n c t i o n . therefore,  of 40-50% of  the  enantiomers hemodialysis  -  39  -  i  EXPERIMENTAL  A.  M a t e r i a l s and Chemicals  1.  Column Chromatography A m b e r l i t e 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 . , Acetone - Reagent g r a d e , F i s h e r S c i e n t i f i c , Methanol - HPLC g r a d e , F i s h e r  Vancouver, B . C . , Canada.  Scientific.  E t h y l a c e t a t e - B u r d i c k & Jackson L a b r a t o r i e s 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  2.  Inc.,  collector,  Muskegon, M i . ,  Sweden.  T h i n - l a y e r Chromatography 1,3-dihydroxynaphthalene Milwaukee, W i . , U . S . A .  (Naphthoresorcinol)  - A l d r i c h Chemical C o . ,  P h o s p h o r i c a c i d (85%) ' - Reagent g r a d e , A l l i e d M i s s i s s a u g a , O n t . , Canada.  Chemical Company,  Whatman K C i s F p l a t e s - Whatman I n c . ,  Nj.,  Clifton,  U.S.A.  S i l G-25 UV25I+ p r e - c o a t e d p l a t e s - Brinkman Instruments Ltd., Rexdale, O n t . , Canada. M e t h a n o l , A c e t o n i t r i l e , Chloroform Scientific.  HPLC g r a d e ,  E t h y l acetate - Burdick & Jackson Laboratories Tetrahydrofuran, Acetic A c i d , T o r o n t o , O n t . , Canada. TLC s t r e a k i n g College, Pa.,  3.  U.S.A.  (Canada)  Fischer  Inc.  Sodium C h l o r i d e - BDH (Canada) L t d . ,  apparatus - A p p l i e d Science U.S.A.  Laboratories  Ltd.,  State  High—performance L i q u i d Chromatography 5 u Ultrasphere I n c . , Berkeley,  ODS column (25 Ca., U.S.A.  Water, A c e t o n i t r i l e , n-Hexane, Scientific.  cm x 4.6  mm), Beckman Instruments  Dichloromethane - HPLC g r a d e ,  Fisher  -  Gas-liquid  40 -  Chromatography  Heptafluorobutyric I I . , USA.  anhydride  - Pierce  Chemical Company,  E t h e r - reagent g r a d e , A l d r i c h Chemical Dimethyl  sulfoxide - Fisher  Sodium h y d r i d e , Company.  Rockford,  Company.  Scientific.  Calcium h y d r i d e ,  D-Glucuronic A c i d , p-Nitrophenol St. Louis, Mo., U.S.A.  Methyliodide - A l d r i c h  Chemical  G l u c u r o n i d e - Sigma Chemical C o .  C h i r a s i l - V a l ® g l a s s 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) Applied Science. F u s e d - s i l i c a c a p i l l a r y column ( u n c o a t e d ) , A l l t e c h A s s o c i a t e s , Arlington Heights, I I . , U . S . A . Carbowax Silar  20 M, A l l t e c h A s s o c i a t e s .  10 C g l a s s  SP-2330 g l a s s U.S.A. 0V-225  glass  capillary  capillary  capillary  column-coated  i n the l a b o r a t o r y .  column - S u p e l c o ,  column, - A p p l i e d  Inc., Bellefonte,  Pa.,  Science  Animal Surgery Ether  s o l v e n t - USP g r a d e , A l d r i c h Chemical  H e p a r i n - Sigma  Company.  Chemical"company.  P o l y e t h y l e n e t u b i n g (PE.50) - Beckton D i c k i n s o n & C o . , P a r s i p p a n y New Y o r k , N . Y . , U . S . A . S i l a s t i c medical grade tubing - Dow Corning C o r p o r a t i o n Products, Midlands, M i . , U . S . A . Dermasept s k i n c l e a n s e r - Germiphene Canada.  Medical  Company L t d . , B r a n t f o r d , Ont  -  6.  41  -  Miscellaneous 3-Glucuronidase Company.  (bovine  liver)  H y d r o c h l o r i c Acid - F i s h e r  -  (5000 u n i t s / m L ,  Scientific.  Ammonium a c e t a t e - G e n e r a l Chemical D i v i s i o n , C o r p o r a t i o n , New Y o r k , Ny, U . S . A . Sodium h y d r o x i d e - American S c i e n t i f i c U.S.A.  Allied  ct-Bromonaphthalene Ny., U.S.A.  Products,  h y d r o c h l o r i d e (W-49167)  - ICN P h a r m a c e u t i c a l s  3-(2,6-Xylyl)5-methylhydantoin  Chemical & Dye  and C h e m i c a l , P o r t l a n d , O r . ,  Tocainide hydrochloride - A s t r a Pharmaceutical Framingham, M a . , U . S . A . l-Amino-2',6'-acetoxylidide Pharmaceutical Products.  Sigma Chemical  Inc.,  -  Inc.,  Astra  P l a i n v i e w , New Y o r k ,  - Astra Pharmaceutical  Products.  2 , 6 - D i m e t h y l a n i l i n e - A l d r i c h Chemical Company. Carbobenzyloxy-D-alanine Sigma Chemical Co.  and N - t e r t « b u t o x y c a r b o n y l - L - a l a n i n e  -  N,N'-Dicyclohexylcarbodiimide, (-f-)di-p-toluoyl-d-tartaric acid monohydrate, 32% hydrogen bromide i n a c e t i c a c i d - A l d r i c h Chemical Company. Benzene, c h l o r o f o r m , ether - D i s t i l l e d i n G l a s s , L a b o r a t o r i e s L t d . , Georgetown, O n t . , Canada. M o n o e t h y l g l y c i n e x y l i d i d e h y d r o c h l o r i d e (MEGX) P h a r m a c e u t i c a l JProducts.  B.  P r e p a r a t i o n o f Reagents and Stock S o l u t i o n s  1.  T o c a i n i d e H y d r o c h l o r i d e S o l u t i o n ( 1 0 ug/mL) T o c a n i d e h y d r o c h l o r i d e (10 mg) was weighed  dissolved 1 mL of  in d i s t i l l e d this  water  i n a 10 ml v o l u m e t r i c  s o l u t i o n was d i l u t e d  100 mL v o l u m e t r i c  flask.  Caledon  Astra  accurately flask.  to volume w i t h d i s t i l l e d  and  An a l i q u o t water  in a  of  -  2.  l-Amino-2',6'-acetoxylidide  flask.  and d i s s o l v e d  An a l i q u o t  d i s t i l l e d water  3.  -  S o l u t i o n (10 ug/ml)  W-49167 H y d r o c h l o r i d e  accurately  42  h y d r o c h l o r i d e (10 mg) was weighed  i n d i s t i l l e d water  of 1 mL of  this  i n a 10 mL v o l u m e t r i c  s o l u t i o n was d i l u t e d to volume w i t h  i n a 100 mL v o l u m e t r i c  flask.  a-Bromonaphthalene S o l u t i o n (10 ug/mL) a-Bromonaphthalene  (10 mg) was weighed a c c u r a t e l y  i n benzene i n 10 ml v o l u m e t r i c f l a s k .  An a l i q u o t  s o l u t i o n was d i l u t e d to volume w i t h benzene  and  of 1 mL of  dissolved this  i n a 100 mL v o l u m e t r i c  flask.  4.  Monoethylglycinexylidide  (MEGX) H y d r o c h l o r i d e  S o l u t i o n (10 ug/mL  M o n o e t h y l g l y c i n e x y l i d i d e h y d r o c h l o r i d e (10 mg) was weighed accurately flask.  and d i s s o l v e d  An a l i q u o t  d i s t i l l e d water  5.  of  i n d i s t i l l e d water  1 mL of  this  i n a 10 mL v o l u m e t r i c  s o l u t i o n was d i l u t e d to volume w i t h  i n a 100 mL v o l u m e t r i c  flask.  T o c a i n i d e Base S o l u t i o n (10 yg/mL) T o c a i n i d e h y d r o c h l o r i d e (50 mg) was d i s s o l v e d  water  and the s o l u t i o n was made a l k a l i n e  sodium h y d r o x i d e s o l u t i o n . 10 mL each of benzene. evaporated in a  by a d d i t i o n of 1 mL of  T h i s s o l u t i o n was e x t r a c t e d  three  1.0 N  times w i t h  The benzene e x t r a c t s were combined and  under vacuum to d r y n e s s .  desiccator.  i n 5 mL d i s t i l l e d  The m a t e r i a l was d r i e d and  stored  -  43  -  T o c a i n i d e base (10 mg) was weighed a c c u r a t e l y of  benzene  i n a 10 mL v o l u m e t r i c f l a s k .  s o l u t i o n was d i l u t e d w i t h benzene  6.  An a l i q u o t  of  to 100 mL to g i v e  1 mL of  a 10 ug/mL  i n 10 mL this solution.  N a p h t h o r e s o r c i n o l Reagent Naphthoresorcinol  ethanol  C.  and d i s s o l v e d  and 35 mL of  (0.2  g)  was d i s s o l v e d  phosphoric a c i d  i n a mixture  of  165 mL of  (85%).  P r e l i m i n a r y S t u d i e s on C a p i l l a r y Column Gas Chromatography o f Tocainide  1.  Column  1.1  Selection  P r e p a r a t i o n o f 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 fused-silica  capillary  column (50 m x 0.2  mm) was coated  carbowax  20 M by the mercury plug dynamic method (Schomburg  1974)  follows:  as  the c a p i l l a r y  s o l u t i o n of carbowax contained inserted seal  i n t o the s o l u t i o n of  The c o a t i n g  p r e s s u r e at was  reservoir  20 M i n d i c h l o r o m e t h a n e .  a s m a l l pool of mercury.  and the side-arm  source.  coating  of the r e s e r v o i r  a v e l o c i t y of 2 cm per s e c .  f i l l e d w i t h the s t a t i o n a r y  The r e s e r v o i r  phase,  to a n i t r o g e n  When 25% of  the column l e n g t h  the column end was lowered The s t a t i o n a r y  w i t h the mercury plug was allowed to flow through the e n t i r e a constant v e l o c i t y .  column was  through the column by n i t r o g e n  the p o o l of mercury to draw i n a 5 cm p l u g .  the column at  also  phase through a septum-type  was connected  s o l u t i o n was f o r c e d  al.,  was f i l l e d w i t h a 2%  One end of the c a p i l l a r y  the s t a t i o n a r y  et  with  In order  into  phase,  along  l e n g t h of  to avoid a sudden change i n  -  c o a t i n g v e l o c i t y as  the s t a t i o n a r y  column was connected to a b u f f e r  the column, the main meters.  the column, the flow of n i t r o g e n was  to evaporate the s o l v e n t .  n i t r o g e n f o r three h o u r s , gas  phase l e f t  column of approximately 10  When the l a s t drop of mercury l e f t increased  44 -  After  d r y i n g under a stream of  the column was t r a n s f e r r e d  to the oven of  chromatograph and one end of the column was connected to  injection port.  The oven was temperature-programmed  the  the  from 5 0 ° to  200°C  at a r a t e of 1 ° per minute and the helium flow through the column was m a i n t a i n e d at  1 mL per m i n u t e .  hours.  c o n d i t i o n i n g , the performance of the column was  After  by a n a l y s i s  The column was h e l d at 2 0 0 ° C f o r 48 evaluated  of a t e s t - p r o b e mixture c o n t a i n i n g nonane, decane,  dibutylketone,  tetradecane,  dodecane,  cis-propylcyclohexanol,  t r a n s - p r o p y l c y c l o h e x a n o l 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 F u s e d - s i l i c a C a p i l l a r y Column In  and  order to enhance the chromatographic p r o p e r t i e s  to i n c r e a s e  s e n s i t i v i t y of d e t e c t i o n to low nanogram  t o c a i n i d e was converted to i t s follows:  to 1.0  of  tocainide  levels,  h e p t a f l u o r o b u t y r y l (HFB) d e r i v a t i v e  ug of t o c a i n i d e base d i s s o l v e d  i n 200 uL of  were added 30 uL of h e p t a f l u o r o b u t y r i c a n y h y d r i d e .  as  n-hexane  The t i g h t l y capped  tubes were heated at 5 5 ° C f o r 30 minutes i n an aluminum b l o c k (Thermolyne D r i - B a t h ,  Fisher S c i e n t i f i c ) .  The excess reagent and  s o l v e n t were removed under a stream of n i t r o g e n and the r e s i d u e reconstituted gas  i n 200 uL of n-hexane.  chromatographic (GC)  analysis.  A 1-2  was  yL a l i q u o t was used f o r  the  -  45 -  Chromatographic c o n d i t i o n s were as temperature,  240°C;  oven temperature, vent f l o w , flow,  1.3  e l e c t r o n capture d e t e c t o r  180°C;  100 mL/min;  50 m L / m i n . ;  follows:  c a r r i e r gas  0.3  (ECD) temperature,  (helium) flow r a t e ,  i n l e t pressure,  c h a r t speed,  injection 350°C;  1 mL/min;  172.3 k P a , make-up gas  split  (helium)  cm/min.  GC A n a l y s i s o f T o c a i n i d e HFB U s i n g C a p i l l a r y Columns Coated w i t h S i l a r 10 C, OV-225 and SP-2330 Chromatographic c o n d i t i o n s f o r a n a l y s i s  heptafluorobutyrate  2.  were the same as d e s c r i b e d  of  tocainide  before.  D e t e r m i n a t i o n o f Optimum C o n d i t i o n f o r D e r i v a t i s a t i o n In  order to determine the optimum time r e q u i r e d  derivatisation,  six  samples of t o c a i n i d e base (1.0  for  ug each)  prepared i n 10 mL p o l y t e t r a f l u o r o - e t h y l e n e - ( P T F E ) l i n e d  were  screw-capped  c u l t u r e tubes and were heated w i t h 30 uL of h e p t a f l u o r o b u t y r i c anhydride (HFBA) f o r reagent  15, 30, 45,  and s o l v e n t  60,  75 and 90 minutes at 5 5 ° C .  (hexane) were removed as  The excess  described before.  r e s i d u e was d i s s o l v e d i n 200 uL of n-hexane c o n t a i n i n g 0.2 a-bromonaphthalene and 1-2  uL of t h i s  carbowax 20 M f u s e d - s i l i c a  c a p i l l a r y column.  against  time of d e r i v a t i s a t i o n  derivatisation.  The  ug of  s o l u t i o n was i n j e c t e d onto a  revealed  A p l o t of peak area  the optimum d u r a t i o n of  ratios  - 46 -  3.  Selection of Internal  Standard  a-Bromonaphthalene was used as e a r l y development of a g a s - l i q u i d  an i n t e r n a l standard  chromatographic assay u s i n g  packed-column and e l e c t r o n capture d e t e c t i o n 1978). it  Although t h i s  technique because of i t s similarity of  HFBA to y i e l d i t s  an i n t e r n a l standard  very short  to t o c a i n i d e .  l i d o c a i n e which i s  (Venkataramanan et  compound had good e l e c t r o n c a p t u r i n g  was not found s u i t a b l e as  d u r i n g the  properties,  i n the present  r e t e n t i o n time and l a c k of any  M o n o e t h y l g l y c i n e x y l i d i d e (MEGX), a  structurally  related  monoheptafluorpbutyrate  to t o c a i n i d e , derivative.  4.  standard  f o r the i n i t i a l  metabolite  reacts  with  T h i s compound has  a l o n g e r r e t e n t i o n time than t o c a i n i d e HFB and was chosen as internal  al.,  the  studies.  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 T o c a i n i d e and I n t e r n a l S t a n d a r d , M o n o e t h y l g l y c i n e X y l i d i d e (MEGX) Compounds w i t h w i d e l y v a r y i n g m o l e c u l a r weights  are known to be d i f f e r e n t i a t e d port for  the s p l i t  mode of  by the g l a s s i n s e r t  injection.  heptafluorobutyryl silica  derivatives  were analysed  c a p i l l a r y column by v a r y i n g the s p l i t  A c a r r i e r gas throughout.  flow r a t e of  used i n the  To t e s t f o r any  between t o c a i n i d e and the i n t e r n a l standard  and v o l a t i l i t y injection  differentiation  (MEGX), a mixture of  their  on a carbowax 20 M fused ratios  from 1:15  to  1:130.  1 mL/min through the column was maintained  -  D.  w i t h an average weight  to 378 j g ) , . were used f o r intravenous  tocainide  Animal Care U n i t of were maintained daylight)  for  chow was fed prior  to,  in this  in metallic  at  to the r a t s .  (216  the pharmacokinetics  (Rats were o b t a i n e d  from the The  animals  cages i n a c o n t r o l l e d environment (12  They were f a s t e d f o r  of  hours  Rat chow or  a p e r i o d of 8-10  lab  hours  Water was allowed ad l i b i t u m .  Plasma L e v e l Study  developed  plasma l e v e l study  of  tocainide  i n the r a t  was c a r r i e d  v e i n cannula f o r blood s a m p l i n g .  used was a minor m o d i f i c a t i o n of  by Weeks and Davis  the  out  The technique  (1964).  P r e p a r a t i o n o f J u g u l a r V e i n Cannula The  joined needle.  jugular  of  polyethylene l e n g t h of  over the metal connector  polyethylene silk  tubing  covered metal  tubing.  i n the  The s i l a s t i c  and p o s i t i o n e d  (PE-50)  hypodermic  about 4 mm l o n g , was i n s e r t e d  t u b i n g to a d i s t a n c e of about 2 mm.  p l a c e by 4-0 The  of  t u b i n g by means of a short  The hypodermic n e e d l e ,  then threaded  section  v e i n cannula c o n s i s t e d  to a s i l a s t i c  polyethylene  "U"  of  l e a s t 3 days p r i o r to the e x p e r i m e n t s .  c a n n u l a t i o n procedure  in  species  study  of about 250 ,g  the U n i v e r s i t y of B r i t i s h C o l u m b i a ) .  u s i n g an implanted j u g u l a r  was  the i n i t i a l  and d u r i n g the e x p e r i m e n t s .  The  1.1  -  P h a r m a c o k i n e t i c s o f T o c a i n i d e i n t h e Rat Adult Wistar r a t s ,  1.  47  over  The s i l a s t i c was  tubing the secured  thread.  cannula end of  the p o l y e t h y l e n e  t u b i n g was  then shaped i n t o a  by bending around a p a s t e u r p i p e t t e and d i p p i n g momentarily  into  - 48 -  b o i l i n g water.  The cannula was cleaned w i t h d i s t i l l e d water,  95% a l c o h o l f o r 3-5  hrs and s t o r e d  i n s t e r i l e normal s a l i n e  prior  to  1.2.  S u r g i c a l I m p l a n t a t i o n o f t h e Cannula rat  was a n e s t h e t i s e d w i t h ether  removing the r a t throughout  from the d e s i c c a t o r ,  the i m p l a n t a t i o n procedure  c o t t o n c l o s e to the nose of  the r a t .  c l i p p e d and a p o i n t was marked at  to i n d i c a t e placed jugular  solution  use.  The  was  soaked i n  the e x t e r i o r i z a t i o n  on i t s  in a desiccator  the a n e s t h e s i a was by p l a c i n g  jar.  maintained  ether-impregnated  The h a i r on t h e ' b a c k the c e n t e r  point for  of  the c a n n u l a .  rapid pulsation  of  T h i s area was r e c o g n i z e d  the j u g u l a r  vein.  The shaved  of  the  the back of The r a t  back and the h a i r on the area over the r i g h t  v e i n was c l i p p e d .  After  animal  the neck was  then  external  by o b s e r v i n g  the  area was c l e a n s e d  by  wiping w i t h a s t e r i l e gauze s a t u r a t e d w i t h 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 l o n g , was made on the over the j u g u l a r surrounding suture.  v e i n and the v e i n was exposed by d e b r i d i n g  fascia.  The v e i n was l i g a t e d  A probe was i n s e r t e d  at  subcutaneously  s a l i n e (20 u n i t s / m L ) ,  j u s t behind the ear  located muscle.  i n the c o r r e c t  plane,  the  The c a n n u l a ,  was then i n s e r t e d  and the probe was p u l l e d towards the j u g u l a r  vein.  ' U ' portion l a i d  If flat  A probe was then placed under the v e i n , r a i s e d  s m a l l puncture was made i n t o the v e i n w a l l .  the  the upper end u s i n g a  the i n c i s i o n on the neck to the back s k i n i n c i s i o n . with heparinised  skin  The t i p of  i n t o the  silk  from filled probe  the cannula was on the neck slightly the  and a  silastic  - 49 -  t u b i n g was cut i n t o a 4 5 ° b e v e l so as  to f a c i l i t a t e  the cannula i n t o the v e i n .  of  h o l e was f u r t h e r a pair  facilitated  of f i n e f o r c e p s  w i t h a slow, advanced  Insertion  easy i n s e r t i o n of  the cannula i n t o the  by g e n t l y l i f t i n g the l i p of  and i n s e r t i n g  puncture  the h o l e w i t h  the cannula i n t o the blood v e s s e l  g e n t l e r o t a t i o n of the t i p .  The s i l a s t i c  t u b i n g was  s l o w l y 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  and then secured  i n the v e i n w i t h a 4-0  silk  suture.  then  tubing  The cannula was  anchored i n p o s i t i o n by t y i n g the suture (used to secure the tubing i n the v e i n )  to the a d j a c e n t muscle.  was r e u n i t e d w i t h s u r g i c a l a 0-0  silk  thread.  subcutaneously closing  The subcutaneous c o n n e c t i v e  thread and the s k i n i n c i s i o n was c l o s e d  The p r o t r u d i n g end of the c a n n u l a was  to the back of  the neck and secured  the s k i n i n c i s i o n w i t h 0-0 s i l k .  ensure that protrude  i t was f u n c t i o n a l .  from the nape of  a p i n i n t o the open end.  mL  i n p o s i t i o n by  About 4 cm of cannula was allowed  to  the neck.  The cannula was s e a l e d by  P e r i o d i c use  of h e p a r i n i s e d  saline  (20 u n i t s / m L ) was taken i n a 1  mL of blood was withdrawn from the  vein cannula.  solutions  h y d r o c l o r i d e ( i n normal s a l i n e )  15,  20 or 25 mg/kg were i n j e c t e d  (20  Collection  animal v i a the j u g u l a r tocainide  inserting  functional.  mL of h e p a r i n i z e d s a l i n e  s y r i n g e w i t h a 23 G needle and 0.2  of  then brought  to  Drug A d m i n i s t r a t i o n and S e r i a l Blood About 0.2  using  The cannula was checked  u n i t s / m L ) helped to keep the cannula  1.3  tissue  Subsequently,  through the j u g u l a r  c a n n u l a was then f l u s h e d w i t h the contents  of  0.5  mL of  aqueous  corresponding  vein cannula.  the f i r s t  syringe  to The  (0.2 mL  -  blood + 0.2  mL h e p a r i n i s e d s a l i n e ) ,  r e c e i v i n g the e n t i r e  180,  240,  heparinised the f l u i d about 0.25 (contents reinjected  300,  thus the r a t was ensured of  dose.  At a p p r o p r i a t e 150,  50 -  time i n t e r v a l s  360,  420,  (3,  6,  10,  15, 20,  120,  s a l i n e was i n t r o d u c e d i n t o the cannula and about 0.15 mL of  was withdrawn.  A f r e s h needle and s y r i n g e was i n t r o d u c e d and  mL of blood was withdrawn. from c a n n u l a , some i n t o the  animal.  The s o l u t i o n i n the f i r s t  blood and h e p a r i n i s e d s a l i n e )  tubes were c e n t r i f u g e d  f o r 10 minutes at  using a  syringe  was  The blood samples were t r a n s f e r r e d  separated plasma samples were kept  2.  90,  480 m i n . ) a s y r i n g e w i t h 0.2 mL of  h e p a r i n i s e d Caraway® tubes and one end was sealed The  30, 45,  to  Critocap®.  2500 r . p . m . and the  frozen u n t i l  analysed.  Urinary Excretion Studies: T o c a i n i d e - d o s e d animals were housed i n s t a i n l e s s  cages with f a c i l i t i e s After  f o r u r i n e c o l l e c t i o n free  steel  of f e c a l  metabolism  contamination.  24 h o u r s , the s i d e s of the cage were washed with d i s t i l l e d water  i n t o an amber c o l o r e d u r i n e c o l l e c t i o n b o t t l e . the u r i n e sample  Suitable  thus c o l l e c t e d were made and kept  frozen  d i l u t i o n s of until  analysed.  E.  D e t e r m i n a t i o n o f C a l i b r a t i o n Curve D a t a and P r e c i s i o n f o r Rat Plasma and U r i n e A s s a y To  five  100 uL a l i q u o t s  of r a t  plasma or u r i n e o b t a i n e d from  u n t r e a t e d male Wistar r a t s , were added 50,  100,  200,  500,  and 1000 ng of  -  51  -  t o c a i n i d e h y d r o c h l o r i d e from an aqueous a l i q u o t of the i n t e r n a l standard m o n o e t h y l g l y c i n e x y l i d i d e (10.0 by 200 pL of  1.0  solution equivalent  pg/mL). to 1.0  T r i p l i c a t e samples of  An  pg of  pg/mL) was added to each t u b e ,  N sodium h y d r o x i d e .  c o n c e n t r a t i o n were p r e p a r e d .  s o l u t i o n (1.0  followed  each  F i v e m i l l i l i t e r s of dichloromethane were  added to each tube and the tubes were shaken f o r 10 minutes on a r o t a r y shaker.  After  c e n t r i f u g a t i o n at  o r g a n i c phase were t r a n s f e r r e d screw-capped  centrifuge  i n a water both at residue  2500 rpm f o r  10 m i n u t e s , 4 mL of  to 15 mL p o l y t e t r a f l u o r o e t h y l e n e  tubes and the c o n t e n t s of the tubes  4 0 ° C under a g e n t l e  stream of n i t r o g e n .  lined  evaporated To the  were added 100 uL of n-hexane and 30 pL of h e p t a f l u o r o b u t y r i c  anhydride.  The tubes were t i g h t l y capped and heated at  minutes i n an aluminum b l o c k .  The excess reagent  200 pL of n-hexane; chromatographic  1-2  pL of t h i s  5 5 ° C f o r 30  was removed by slow  e v a p o r a t i o n w i t h a stream of n i t r o g e n and the r e s i d u e  F.  the  was d i s s o l v e d  s o l u t i o n was used f o r  in  gas  analysis.  R e s o l u t i o n o f T o c a i n i d e Enantiomers on C h i r a s i l - V a l  8  Glass  Capillary  Column To  1.0  screw-capped  pg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e c o n t a i n e d i n 10 mL c u l t u r e tubes (0.1  pg/mL) were added 200pL of 1.0 dichloromethane. a rotary  shaker.  mL of a stock  s o l u t i o n c o n t a i n i n g 10  N sodium hydroxide s o l u t i o n and 5 mL of  The tubes were t i g h t l y capped and shaken f o r The dichloromethane was separated  c u l t u r e tube and the contents were evaporated  10 min on  to a c l e a n , d r y  to dryness  under a  stream  -  of  nitrogen.  contents  of  To the dry r e s i d u e  -  was added 200 uL of n-hexane  the tube were mixed by v o r t e x i n g  H e p t a f l u o r o b u t y r i c anhydride (30 was  52  uL) was  f o r a few seconds.  then added to the tube and  t i g h t l y capped w i t h a p o l y t e t r a f l u o r o e t h y l e n e  and heated  at  1 \iL of  s o l u t i o n was i n j e c t e d  c a p i l l a r y column.  chromatograph  equipped w i t h a s p l i t  mode i n j e c t i o n port  detector  the chromatographic  and a  S y n t h e s i s o f T o c a i n i d e Enantiomers  1.  S y n t h e s i s o f R(-) T o c a i n i d e H y d r o c h l o r i d e  and 7.3  g (0.06  mole)  g (0.06  (Hewlett  Ni  Packard)  electron  capture  mole)  of  carbobenzyloxy-D-alanine  of 2 , 6 - d i m e t h y l a n i l i n e i n 150 ml of d i c h l o r o -  methane were added 13.6 in  13.38  6 3  glass  analysis.  G.  To a s o l u t i o n of  tube was  and  onto the C h i r a s i l - V a l ®  A model 5830A gas  was used f o r  screw-cap  the odour of HFBA was not  To the r e s i d u e was added 200 uL of n-hexane this  The  it  and the excess reagent was removed by  e v a p o r a t i o n w i t h a stream of n i t r o g e n u n t i l apparent.  (PTFE)-lined  5 5 ° C i n an aluminum b l o c k f o r 30 m i n u t e s .  c o o l e d to room temperature  and the  g (0.066 mole)  60 mL of d i c h l o r o m e t h a n e .  After  of N , N ' - d i c y c l o h e x y l c a r b o d i i m i d e  the s o l u t i o n was l e f t  standing  at  room temperature  f o r one h o u r , the p r e c i p i t a t e d N , N ' - d i c y c l o h e x y l u r e a  was f i l t e r e d  and the s o l v e n t was evaporated  off  reduced p r e s s u r e l e a v i n g 12.3  g of white s o l i d , m.p.  remove the carbobenzyloxy group, bromide i n a c e t i c  from the f i l t r a t e 167-168°C.  under To  70 mL of a s o l u t i o n of 30-32% hydrogen  a c i d were added to 12.3  g of  the above  reaction  -  product and the mixture was s t i r r e d  53  -  until  dissolved.  To t h i s  200 ml of dry d i e t h y l e t h e r were added and the p r e c i p i t a t e d hydrobromide was f i l t e r e d m.p. 2 6 7 ° C .  off  and d r i e d , y i e l d i n g 8.2  T h i s m a t e r i a l was converted  265-266°C material  to y i e l d R(-)  and ( a ) p 5 - 4 2 . 1 6 ° was converted  anhydride as  described  chromatographic synthesized  (c,  to i t s  2.63  to a h y d r o c h l o r i d e s a l t w i t h  i n methanol). with  from  A  sample of  ratio  column.  of 95:5  this  heptafluorobutyric  The p u r i t y was determined by  a n a l y s i s on a C h i r a s i l - V a l ®  had an enantiomer  solid,  t o c a i n i d e h y d r o c h l o r i d e , m.p.  derivative  before.  tocainide  g of white  e t h a n o l i c hydrogen c h l o r i d e s o l u t i o n and r e c r y s t a l l i z e d e t h a n o l - d i e t h y l ether  solution,  for  gas  The product  the R and S  enantiomers,  respectively.  2.  S y n t h e s i s o f S(+) T o c a i n i d e H y d r o c h l o r i d e To a s o l u t i o n of  alanine  and 12.1  g (0.1  18.9  g (0.1  mole)  hour,  After  g (0.1  the p r e c i p i t a t e d  of at  N,N'-dicyclohexylroom temperature  N , N ' - d i c y c l o h e x y l urea was f i l t e r e d from the f i l t r a t e  gm of a creamy white s o l i d , m.p. To remove  mole)  the mixture was s t i r r e d  s o l v e n t was evaporated 16.1  of N - t e r t * b u t o x y c a r b o n y l - L -  of 2 , 6 - d i m e t h y l a n i l i n e i n 200 ml of  dichloromethane were added 20.6 carbodimide.  mole)  stirred  filtered  the  leaving  until  50 mL of 30-32%  to 10 g of dissolved.  the above To t h i s  200 mL of dry d i e t h y l e t h e r were added and the p r e c i p i t a t e d hydrobromide was  and  131°C.  a c i d were added  product and the mixture was  off  under reduced p r e s s u r e ,  the N - t e r t • b u t o x y c a r b o n y l group,  hydrogen bromide i n a c e t i c  for 1  and d r i e d , y i e l d i n g 7.8  reaction  solution  tocainide  g of white  solid,  -  m.p.  274°C.  A sample of  heptafluorobutyryl derivative to c o n s i s t  p u r i t y of  this  of 81:19  product,  ratio  of  t o c a i n i d e b a s e , obtained  tartaric  a c i d i n 10 mL of 95% e t h a n o l .  material  to a warm s o l u t i o n of  at  was converted  ethanol-diethyl  this material  S:R i s o m e r s .  was added  5 times  ether  to y i e l d S(+)  To i n c r e a s e the from 1 g o f  optical  the  g of d i - p - t o l u o y l - 1 salt  to y i e l d f i n e n e e d l e s .  hydrochloride salt  to y i e l d S(+)  as  column and was  The d i a s t e r e o i s o m e r i c  room temperature to i t s  1.4  tocainide was analysed  on the C h i r a s i l - V a l ®  material  crystallized  to a h y d r o c h l o r i d e s a l t and  from e t h a n o l - d i e t h y l ether  h y d r o c h l o r i d e , m.p. 2 6 6 ° C .  found  -  T h i s m a t e r i a l was converted  recrystallized  its  54  was  This  and r e c r y s t a l l i z e d  t o c a i n i d e h y d r o c h l o r i d e , m.p.  from  266°C,  25  [ct]  +42.35°  D  ratio  of  indicated 3.  (c  2.63  i n methanol).  An e v a l u a t i o n of  the h e p t a f l u o r o b u t y r y l d e r i v a t i v e a ratio  o f 91:9  of the S:R  the  enantiomer  on the C h i r a s i l - V a l ® column  isomers.  D e t e r m i n a t i o n o f o p t i c a l p u r i t y and I d e n t i t y o f S(+) and R ( - ) t o c a i n i d e by gas chromatograpby/mass s p e c t r o m e t r y (GCMS) In order  to determine  the c o m p o s i t i o n of the product  synthesized,  a GCMS technique u s i n g C h i r a s i l - V a l ® g l a s s c a p i l l a r y was employed. The GCMS a n a l y s i s of the h e p t a f l u o r o b u t y r y l d e r i v a t i v e s tocainide  was done under the f o l l o w i n g c o n d i t i o n s :  temperature,  240°C;  oven temperature,  to 2 0 0 ° C at 5 ° C / m i n ; 200°C;  analyser  s p l i t l e s s mode of  temperature,  ionisation potential,  70  ev.  240°C;  transfer  (R,S)-  injection  5 0 ° C to 1 5 0 ° C at injection,  of  30°C/min,  source  150°C  temperature,  l i n e temperature,  240°C,  - 55 -  4.  Measurement o f O p t i c a l R o t a t i o n o f T o c a i n i d e Enantiomers Optical  r o t a t i o n s t u d i e s were conducted on a P e r k i n - E l m e r Model  142 p o l a r i m e t e r 25°C.  ( P e r k i n - E l m e r , Norwa^lk, C t . , USA) i n a  T o c a i n i d e h y d r o c h l o r i d e (131.5 mg) was a c c u r a t e l y  dissolved  i n 5 mL of methanol (HPLC g r a d e ) .  was f i l l e d w i t h t h i s The  0.1  specific  The c e l l  s o l u t i o n and the o p t i c a l  dm tube at  weighed and  of the  r o t a t i o n was  r o t a t i o n of S ( + ) t o c a i n i d e h y d r o c h l o r d e ( c ,  polarimeter  measured.  2.63%  i n MeOH)  25 was  (a)D  = (+)  42.35°  hydrochloride (c,  H.  1.  and the s p e c i f i c  2.63  Determination  r o t a t i o n of R(-)  i n MeOH) was ( a ) "  = (-)  tocainide  42.16°.  o f C a l i b r a t i o n Curve Data and P r e c i s i o n o f A s s a y  o f T o c a i n i d e Enantiomers i n Human Plasma and U r i n e To 0.5  mL of plasma were added  ng  of racemic  tocainide  of  the i n t e r n a l s t a n d a r d .  2000 ng of racemic  2.  prepared  In a s i m i l a r  100 uL of pg/mL),  fashion  t o c a i n i d e were added to 0.1  were e x t r a c t e d  I n t e r - and I n t r a - A s s a y To 0.1  250,  750,  1000,  and  s o l u t i o n and 1000 ng  100, mL of  200,  400,  1000,  and  u r i n e along w i t h  and d e r i v a t i s e d  as  described  before.  Variations i n three c u l t u r e t u b e s , were  hydrochloride solution (stock s o l u t i o n ,  10 ug/mL), 200 uL of  1.0  added  10  100 uL of m o n o e t h y l g l y c i n e x y l i d i d e h y d r o c h l o r i d e s o l u t i o n  solution,  1500  T r i p l i c a t e samples of plasma and u r i n e  mL of u r i n e , c o n t a i n e d  (±)tocainide  500,  h y d r o c h l o r i d e from a stock  1000 ng of i n t e r n a l s t a n d a r d . thus  100,  N sodium hydroxide s o l u t i o n and  (stock  -  5 mL of d i c h l o r o m e t h a n e . tube  30  The dichloromethane was separated  into  tubes and the s o l v e n t was removed by e v a p o r a t i o n  w i t h a stream of n i t r o g e n . and  -  The tubes were t i g h t l y capped and tumbled on a  tumbler f o r 10 m i n u t e s .  three c l e a n dry c u l t u r e  56  To the r e s i d u e were added 200  pL of h e p t a f l u o r o b u t y r i c a n h y d r i d e .  pL of  The tubes were  n-hexane  tightly  capped and heated i n an aluminum block at 5 5 ° C f o r 30 m i n u t e s .  The  excess reagent was removed by slow e v a p o r a t i o n at room temperature a stream of n i t r o g e n and the r e s i d u e was r e c o n s t i t u t e d n-hexane.  An a l i q u o t of 1 pL of  Chirasil-Val® internal  standard were determined.  between i n j e c t i o n s  I.  s o l u t i o n was i n j e c t e d  pL of  onto  g l a s s c a p i l l a r y column and the peak area r a t i o s  tube and the peak area r a t i o s  these  this  i n 200  of  with  the  of drug  to  Three i n j e c t i o n s were made from each  measured were used  the same sample as w e l l  to compute v a r i a t i o n s  as v a r i a t i o n s  between  samples.  P r e l i m i n a r y Study o f S t e r e o s e l e c t i v i t y o f T o c a i n i d e D i s p o s i t i o n i n the Human Two  kg),  fasted  h e a l t h y male v o l u n t e e r s overnight for  (38 y e a r s ,  68 kg and 39 y e a r s ,  98  12 h , were a d m i n i s t e r e d 100 mL of an o r a l  s o l u t i o n of racemic t o c a i n i d e h y d r o c h l o r i d e at a dose of 3 mg/kg body weight under the s u p e r v i s i o n of a p h y s i c i a n .  Food was allowed  three hours and water was allowed _ad l i b i t u m . cannula was i n s e r t e d collect  i n the c u b i t a l v e i n of  blood samples f o r  the f i r s t  were c o l l e c t e d by v e n i p u n c t u r e .  3 hours.  An i n d w e l l i n g  after  butterfly  the arm and was used Subsequent  to  blood samples  Blood samples (8 mL) were c o l l e c t e d  at  -  15 minute i n t e r v a l s hours t h e r e a f t e r . plasma was s t o r e d in  for  the f i r s t  57 -  2 hours and at  3,  Blood samples were c e n t r i f u g e d at - 2 0 ° C u n t i l  p o l y e t h y l e n e bags at 0,  1,  2,  assayed. 3,  5,  5,  7,  and the  24,  48,  and 72  separated  U r i n e samples were c o l l e c t e d  and 7 hours and t h e r e a f t e r  at  the  convenience of the s u b j e c t and at blood withdrawal times up to 96 hours.  Samples  were kept f r o z e n u n t i l  analysed.  Samples  of plasma and  u r i n e were a l s o o b t a i n e d before drug i n g e s t i o n to serve as blanks and for  the d e t e r m i n a t i o n of c a l i b r a t i o n curve data  J.  1.  and p r e c i s i o n .  Comparison o f t h e A n a l y t i c a l R e s u l t s O b t a i n e d f o r Racemic T o c a i n i d e U s i n g 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 w i t h those o f T o c a i n i d e Enantiomers U s i n g t h e Chirasil-Val® G l a s s C a p i l l a r y Column In  racemate,  order to compare the v a l u e s  of the  total  the same samples from one v o l u n t e e r were a l s o analysed by a  non-stereospecific fused-silica  2.  obtained by a n a l y s i s  c a p i l l a r y column gas  chromatographic method employing  c a p i l l a r y column (50 m x 0.2  mm) coated w i t h carbowax 20 M.  D e t e r m i n a t i o n o f C a l i b r a t i o n Curve Data and P r e c i s i o n o f Human Plasma A s s a y 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 ( ± )  h y d r o c h l o r i d e along with 1000 ng of i n t e r n a l standard added, were e x t r a c t e d , The  relative  derivatised  and analysed  (MEGX) had been  as d e s c r i b e d  standard d e v i a t i o n of the peak area r a t i o s  samples of each c o n c e n t r a t i o n were d e t e r m i n e d .  tocainide  for  before. triplicate  -  58 -  K.  Determination  o f C a l i b r a t i o n Curve Data and A s s a y P r e c i s i o n  1.  Human Plasma and U r i n e 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 I n t e r n a l Standard To 0.5  ng  1-aminoacetoxylidide.  derivatised  and analysed  These samples were  as d e s c r i b e d  the i n t e r n a l  extracted,  before.  C o n f i r m a t i o n o f the S t r u c t u r e o f the A c y l D e r i v a t i v e o f 1-aminoacetoxylidide To  1.0  The mixture was shaken f o r 10 minutes on a  The dichloromethane was s e p a r a t e d ,  derivatised  evaporated  residue  before.  u s i n g GCMS and both e l e c t r o n impact ( E l )  i o n i z a t i o n (CI)  tube  and the  w i t h h e p t a f l u o r o b u t y r i c anhydride as d e s c r i b e d  sample was analysed chemical  s o l u t i o n were  1.0 N sodium hydroxide s o l u t i o n and 5 mL of  dichloromethane. tumbler.  (W-^49167) by C a p i l l a r y GCMS  pg of W-49167 h y d r o c h l o r i d e i n aqueous  added 200 pL of  L.  mL of blank u r i n e were added 25  to 2000 ng of ( ± ) t o c a i n i d e h y d r o c h l o r i d e and 1000 ng of  standard,  2.  mL of blank plasma or 0.1  This  and  s p e c t r a were o b t a i n e d .  E f f e c t o f Sodium Hydroxide Treatment on U r i n e C o n t a i n i n g T o c a i n i d e Carbamoyl-OIt  has  fr-D-glucuronide  been r e p o r t e d  alkaline solutions  and that  that e s t e r g l u c u r o n i d e s are u n s t a b l e they are r e a d i l y h y d r o l y s e d thus  the parent compound (Hasegawa  et a l . ,  1982).  Since  i n v o l v e d a d d i t i o n of sodium hydroxide s o l u t i o n (200 solution)  to l i b e r a t e  the f r e e  base from i t s  salts  in  generating  the assay procedure pL of 1.0 N present  i n the u r i n e ,  -  it  was important to e s t a b l i s h  59 -  that no t o c a i n i d e was generated  g l u c u r o n i d e d u r i n g the assay p r o c e d u r e . an i n t e r n a l standard  t i o n of  t o c a i n i d e i n u r i n e u s i n g a s h o r t SE-30 (methyl  detection. retention  i n t h i s experiment f o r  c a p i l l a r y column (15 m x 0.25 T o c a i n i d e , as  the f r e e  silicone)  mm) w i t h flame  base, e l u t e d  was  the GLC-determina-  from t h i s  time of 4 minutes and the i n t e r n a l standard  minutes (column temperature  its  Pure diphenhydramine (base)  used as  fused-silica  from  ionisation column w i t h a  e l u t e d at  2.6  160°).  Procedure An a l i q u o t of 0.1  mL of u r i n e c o n t a i n i n g h i g h p r o p o r t i o n s of  t o c a i n i d e g l u c u r o n i d e (15 hour u r i n e sample from one of was dispensed -  13.0)  i n t o 5 t e s t tubes and 0.5  was added  to each tube.  mL of a buffer  A buffer  the  volunteers),  s o l u t i o n (pH, 7.0  s o l u t i o n (0.2  M) c o n t a i n i n g  b o r i c a c i d , potassium c h l o r i d e , and sodium hydroxide was used the pH of  the u r i n e .  The contents were e x t r a c t e d  d i c h l o r i d e and evaporated  to dryness  by a g e n t l e  to a d j u s t  with 5 mL of methylene stream of n i t r o g e n .  To  the r e s i d u e were added 50 uL of diphenhydramine s o l u t i o n (1 mg/mL i n CH3OH) and the samples were mixed on a v o r t e x m i x e r . uL of  this  following = 160°C; mL/min;  s o l u t i o n was used f o r gas conditions:  c a r r i e r gas make-up gas  chromatographic a n a l y s i s  i n j e c t i o n temperature = 2 4 0 ° C ; (He) flow r a t e = 1 mL/min;  (He) = 50 mL/min.  An a l i q u o t of  column  under  1-2 the  temperature  s p l i t vent flow = 30  -  M.  Pharmacokinetics  1.  Intravenous  o f T o c a i n i d e Enantiomers i n H e a l t h y  fasted  for  R,S-tocainide  (age,  38-42 y e a r s ;  12 hours o v e r n i g h t , were a d m i n i s t e r e d  were recorded p r i o r t o ,  and 1 and 2 hours a f t e r  food was allowed f o r 3 hours p o s t - i n f u s i o n . collected into heparinized V a c u t a i n e r s ®  1.0,  1.5,  saliva  cannula i n s e r t e d  2.0,  3,  5,  7,  10,  72.0  and 96.0  The pH of  immediately a f t e r for  the drug i n f u s i o n .  analysis.  1.0,  hours and at  (Becton D i c k i n s o n & C o . ) through  48,  and 72 h o u r s .  2.0,  collection.  3.0,  5.0,  enantiomers  as  its  7.0,  volume was  U r i n e was s t o r e d  The thawed u r i n e was c e n t r i f u g e d  described  0.75,  Unstimulated at  the same  Urine  10.0,  24.0,  at - 2 0 ° C u n t i l at  other  measured required  2000 r . p . m . f o r  minutes and a 100 uL a l i q u o t was used f o r e x t r a c t i o n of  2.  0.5,  the convenience of the s u b j e c t at  the u r i n e as w e l l as  No  Blood samples (8 mL) were  blood samples and the pH was immediately measured.  samples were a l s o c o l l e c t e d at  times.  30,  a r a t e of 10  Electrocardiograms  i n the b r a c h i a l v e i n at 0 . 3 , 24,  65-98  200 mg of  samples (2 mL) were c o l l e c t e d i n t o 5 mL g l a s s v i a l s  time as  48.0,  weight,  h y d r o c h l o r i d e i n t r a v e n o u s l y as an i n f u s i o n at  mg/min f o r 20 minutes under m e d i c a l s u p e r v i s i o n .  a butterfly  Subjects  A d m i n i s t r a t i o n o f Racemic T o c a i n i d e H y d r o c h l o r i d e  F i v e h e a l t h y male v o l u n t e e r s kg)  60 -  10  tocainide  before.  O r a l A d m i n i s t r a t i o n o f Racemic T o c a i n i d e H y d r o c h l o r i d e Tocainide hydrochloride tablets  h e a l t h y male v o l u n t e e r s  (200 mg) were g i v e n to  who were f a s t e d o v e r n i g h t .  seven  Blood samples (8 mL)  were c o l l e c t e d i n t o h e p a r i n i s e d V a c u t a i n e r s ® through a b u t t e r f l y  cannula  -  inserted 2.0,  i n the b r a c h i a l v e i n at 0 . 2 5 ,  3.0,  collected  5.0, at  hours and at  7.0,  1.0,  collection.  10.0,  2.0,  24.0,  3.0,  36.0,  5.0,  -  0.5,  0.75,  1.0,  as i t s  7.0,  10.0,  volume was measured  Urine was s t o r e d  thawed u r i n e was c e n t r i f u g e d  at at  -20°C  24.0,  48.0,  until  1.5,  1.75,  U r i n e was 72.0  and 96  times.  immediately  The pH o f  after  required for a n a l y s i s .  2000 r . p . m . f o r  was used f o r e x t r a c t i o n of  1.25,  48 and 72 h o u r s .  the convenience o f the s u b j e c t at other  the u r i n e as w e l l  aliquot  61  The  10 minutes and a 100 uL  t o c a i n i d e enantiomers  as  described  before.  N.  1.  Assay o f T o c a i n i d e H y d r o c h l o r i d e T a b l e t s T o c a i n i d e h y d r o c h l o r i d e t a b l e t s were weighed (10 t a b l e t s )  average weight was d e t e r m i n e d .  A powdered sample,  of  t o c a i n i d e h y d r o c h l o r i d e , was d i s s o l v e d  in  a 50 mL v o l u m e t r i c f l a s k .  diluted  which 0.1 analysed  2.  mL (1 yg) was used f o r under i d e n t i c a l  i n d i s t i l l e d water  One m i l l i l i t e r  to 100 mL w i t h d i s t i l l e d water  s o l u t i o n was  T r i p l i c a t e samples were  Injection mL o f the i n j e c t i o n was  out and d i l u t e d to 10 mL with d i s t i l l e d water.  s o l u t i o n (1 mg/mL) a d i l u t e 0.1  mL of t h i s  were analysed  contained  conditions.  From a 5 mL ampoule (50 mg/ml), 0.2  and  of t h i s  to 50 mg  to g i v e a 10 ug/mL s o l u t i o n , from  the a s s a y .  Assay o f T o c a i n i d e H y d r o c h l o r i d e  pipetted  equivalent  and an  From t h i s  s o l u t i o n c o n t a i n i n g 10 ug/mL was  s o l u t i o n was used  for  the a s s a y .  and the area r a t i o s compared to that of  t o c a i n i d e h y d r o c h l o r i d e processed  i n the same manner.  stock  prepared  T r i p l i c a t e samples 1 yg of  standard  -  3.  62  -  D e t e r m i n a t i o n o f A d s o r p t i o n o f T o c a i n i d e by t h e P l a s t i c T u b i n g o f the I n f u s i o n S e t In  order to r u l e out a d s o r p t i o n of the drug by the  t u b i n g used f o r assayed before  the i n f u s i o n s e t , and a f t e r  t o c a i n i d e h y d r o c h l o r i d e i n j e c t i o n was  p a s s i n g through the i n f u s i o n s e t .  time of the s o l u t i o n w i t h the p l a s t i c samples were assayed as  0.  1.  The c o n t a c t  t u b i n g was 20 m i n u t e s .  These  the racemate u s i n g 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). prepared  plastic  Three samples f o r  analysis  were  as d e s c r i b e d under assay of i n j e c t i o n .  Chromatographic A n a l y s i s  o f Uremic Plasma E x t r a c t  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 o b t a i n e d from a p a t i e n t creatinine,  13.4 mg%) was e x t r a c t e d ,  Chirasil-Val® described  2.  fused-silica  who was a n e p h r i c  derivatised  (serum  and analysed on a  c a p i l l a r y column under the c o n d i t i o n s  before.  Chromatographic A n a l y s i s  o f Uremic Plasma E x t r a c t  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 c o n t a i n i n g v a r y i n g amounts of c r e a t i n i n e (4.9 16.8 mg%) ( o b t a i n e d derivatised  from Vancouver General H o s p i t a l ) were  and analysed  column (50 m x 0.2  on a carbowax 20 M f u s e d - s i l i c a  mm) as d e s c r i b e d  before.  extracted, capillary  to  -  3.  63  -  Chromatographic A n a l y s i s o f Uremic Plasma E x t r a c t s on a D u a l C a p i l l a r y Column (Carbowax 20 M and Chirasil-Val®) In  material,  order  to separate i n t e r f e r i n g peaks due to  two columns were connected  (Chromatographic S p e c i a l i t i e s , Plasma described  20 M f u s e d - s i l i c a  side.  of  mg% c r e a t i n i n e were analysed  of a carbowax  column (50 m x 0.2  s i d e and a  fused-silica  mm) on the i n j e c t o r  column (0.50  m x 0.3 for  mm) on the  detector  the a n a l y s i s  of  plasma  from a p a t i e n t who was anephric and who had been g i v e n 200 mg  ( ± ) t o c a i n i d e h y d r o c h l o r i d e by i n t r a v e n o u s  4.  as  employing c a p i l l a r y columns c o n s i s t i n g  T h i s combination of columns was used  collected  by a column-coupler  Brockville, Ontario).  samples c o n t a i n i n g 13.4  before,  Chirasil-Val®  together  endogenous  infusion.  P r e l i m i n a r y Study o f T o c a i n i d e 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  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 Racemic T o c a i n i d e  Hydrochloride  Racemic t o c a i n i d e h y d r o c h l o r i d e was a d m i n i s t e r e d rate intravenous age, mg%.  23 y e a r s ;  i n f u s i o n (10 mg/min)  2 years.  3.0,  5.0,  syringe  had been undergoing h e m o d i a l y s i s  Blood (1 mL) was withdrawn at 0 . 3 3 ,  7.0,  10.0,  12.0  to a p a t i e n t  through a b u t t e r f l y  three  0.5,  and 24 hours p o s t - i n f u s i o n cannula i n s e r t e d  Blood was immediately t r a n s f e r r e d 1000  constant  w e i g h t , 75 kg) w i t h a serum c r e a t i n i n e l e v e l of  This patient  for  f o r 20 minutes  by  to c u l t u r e  (male,  13.4  times a week  0.75,  1.0,  2.0,  into a glass  i n the b r a c h i a l  vein.  tubes and c e n t r i f u g e d  g f o r 10 minutes and the plasma was separated and kept  frozen  at  -  at - 2 0 ° C u n t i l  analysed.  glass v i a l s  the same time as  and  at  64  -  S a l i v a (1 mL) was a l s o c o l l e c t e d i n t o 5 mL plasma c o l l e c t i o n , the pH was  the samples were kept f r o z e n at - 2 0 ° C u n t i l  4.2  required for  the a s s a y .  Hemodialysis Twenty-four hours a f t e r  the i n t r a v e n o u s i n f u s i o n of  t o c a i n i d e h y d r o c h l o r i d e , h e m o d i a l y s i s was s t a r t e d hours u s i n g a h o l l o w f i b e r d i a l y s e r (surface  area 0.8 m ) .  dialysate  (CF 1211)  period  with a cuprophan membrane  The blood flow r a t e was 200 mL/min and the  flow r a t e was 500-550 mL/min.  for  racemic  f o r a p e r i o d of 5  Both a r t e r i a l  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  P.  measured,  the d e t e r m i n a t i o n of t o c a i n i d e  and venous blood  d u r i n g the  dialysis  levels.  D e t e r m i n a t i o n o f C a l i b r a t i o n Curve Data and P r e c i s i o n o f Assay of  Saliva The  r.p.m. clear 100,  for  frozen s a l i v a  10 minutes to remove p a r t i c u l a t e m a t t e r .  supernatant 200,  samples were thawed and c e n t r i f u g e d  400,  at 2000  To 0.5 mL of  the  s a l i v a c o n t a i n e d i n a 10 mL c u l t u r e tube were added  1000,  1500 and 3000 ng of racemic t o c a i n i d e ,  1000 ng of the i n t e r n a l standard  (W-49167).  Subsequently,  along w i t h 200 yL of  1.0 N sodium hydroxide s o l u t i o n and 5 mL of dichloromethane were added and as  the t o c a i n i d e enantiomers were e x t r a c t e d , described  p r e v i o u s l y under a n a l y s i s  derivatised  and analysed  of plasma and u r i n e .  -  65  -  Q.  A n a l y s i s o f Tocainide Metabolites i n the Urine  1.  I s o l a t i o n o f Glucuronides  from U r i n e by A d s o r p t i o n on XAD-2 R e s i n  Column Fifty glass  grams of a m b e r l i t e XAD-2 r e s i n were packed i n a 3 x 60 cm  column.  The column was washed with 200 mL of  500 ml of d i s t i l l e d water.  Subsequently,  acetone,  200 mL o f u r i n e ( o b t a i n e d  a h e a l t h y v o l u n t e e r who had been dosed with 200 mg of ( ± ) hydrochloride) mL/min.  were p e r c o l a t e d  amino a c i d s ,  e l u t e d w i t h 600 mL of methanol.  The e l u a t e was c o l l e c t e d  using a f r a c t i o n c o l l e c t o r .  for  s i l i c a gel  the presence  p l a t e s at taken as  i n ethanol/phosphoric a c i d ,  methanolic f r a c t i o n s  adjusted  s p r a y i n g w i t h 0.1%  followed by h e a t i n g  the  (p-Nitrophenol  i n an i d e n t i c a l manner gave a blue c o l o u r ) .  The  c o n t a i n i n g the g l u c u r o n i d e s were pooled and the  to pH 3 with 1.0  mL p o r t i o n s of hexane.  The aqueous r e s i d u e  was  N - h y d r o c h l o r i c a c i d and washed w i t h t h r e e ,  The aqueous phase was then f r e e z e - d r i e d  was r e c o n s t i t u t e d  chromatography.  i n 5 mL  the sample on a  of g l u c u r o n i d e s .  was removed by vacuum e v a p o r a t i o n .  methanolic extract  and then  The appearance of a b l u e c o l o u r was  an i n d i c a t i o n of the presence treated  to  Every 5th f r a c t i o n was then  of g l u c u r o n i d e s by s p o t t i n g  1 3 0 ° C f o r 10 m i n u t e s .  glucuronide,  solvent  inorganic salts)  t h i n - l a y e r chromatography (TLC) p l a t e ,  naphthoresorcinol  residue  tocainide  The column was then washed with 500 mL of d i s t i l l e d water  fractions  from  through the r e s i n column at a r a t e of 3  remove unbound substances ( u r e a ,  tested  f o l l o w e d by  i n methanol.  was used  for  further  This p a r t i a l l y analysis  purified  by t h i n - l a y e r  60  and the  - 66 -  2.  T h i n - l a y e r Chromatographic S e p a r a t i o n o f the G l u c u r o n i d e s Methanolic extract  described  was a p p l i e d  w i t h a standard  For  the crude u r i n e sample (5  to a TLC p l a t e (Whatman K C i s , 5 x 20 cm) along  Six  such p l a t e s were developed  1.  25% methanol/75%  water  2.  50% methanol/50%  water  3.  60% methanol/40%  water  4.  75% methanol/25%  water  5.  90% tetrahydrofuran/10%  6.  75% a c e t o n i t r i l e / 2 5 %  i n the f o l l o w i n g  water  water  c o n t a i n i n g more than 40% water,  chloride sufficient  to g i v e a a 0.5  an amount of  was allowed  to run to 15 cm on the p l a t e .  w i t h n a p h t h o r e s o r c i n o l reagent and heated solvent  water was  systems  tested,  tanks.  The  The s o l v e n t  The p l a t e s were then sprayed at  1 3 0 ° for  10 mins.  Out of  the combination of 60% methanol 40%  found to be the most o p t i m a l s o l v e n t  crude m e t h a n o l i c u r i n e  sodium  M c o n c e n t r a t i o n was added.  p l a t e s were developed i n 6 x 22 cm c i r c u l a r g l a s s  3.  pL of a 1  systems:  solvents  the s i x  uL) p r e v i o u s l y  g l u c u r o n i d e , p - N i t r o p h e n o l g l u c u r o n i d e (5  mg/mL s o l u t i o n ) . solvent  of  system  for  separating  the  extract.  P r e p a r a t i v e T h i n - l a y e r Chromatographic I s o l a t i o n o f Tocainide  Glucuronides  An a l i q u o t of 150 pL of  the crude m e t h a n o l i c e x t r a c t . w a s  applied  -  67  -  i n a narrow band to s i x 20 x 20 cm KCjg p l a t e s and was developed with methanol/water  (60:40)  and to avoid h e a t i n g  s o l v e n t system.  the whole p l a t e ,  of  labelled,  sprayed with n a p h t h o r e s o r c i n o l reagent  1 3 0 ° for  10 m i n s .  unsprayed p o r t i o n of corresponding of  with a diamond c u t t e r .  The g l a s s the p l a t e  to most i n t e n s e  the p l a t e s ,  and r e - a p p l i e d  strips  were  to l o c a t e  to a second C^g p l a t e  well  resolved  In order  to c o n f i r m  t o c a i n i d e i n the i s o l a t e d  gas  the separated bands. off  Bands  the remainder  from the support m a t e r i a l was  (20 x 20 cm) as a narrow band. (50:50)  s o l v e n t system.  there was no c o n t a m i n a t i o n with pure  The  Two  and l a b e l l e d as band  that bands,  with  concentrated  and 0.69  respectively).  t o c a i n i d e base was a l s o  analysed  the u r i n e sample and the Rp v a l u e was  o b s e r v a t i o n under U . V . l i g h t (Rf = 0 . 5 5 ) .  The crude m e t h a n o l i c e x t r a c t and l b , were  the  0.84  under i d e n t i c a l c o n d i t i o n s as recorded after  of g l a s s were  then a l i g n e d w i t h  from each o t h e r , were i s o l a t e d  l a and band l b (Rf v a l u e s ,  separation  and heated i n an oven  The c l e a r methanolic e x t r a c t  samples were e l u t e d w i t h methanol:water bands,  The s t r i p s  blue c o l o u r were scraped  and these were e x t r a c t e d  methanol and c e n t r i f u g e d .  the band  a 1 cm width of g l a s s was cut from  both ends  at  the p l a t e  To observe  as w e l l  as  the separated bands,  then examined by high-performance l i q u i d  chromatography/mass  spectrometry.  la  chromatography and  -  4.  68  -  M i c r o b o r e LCMS o f T o c a i n i d e G l u c u r o n i d e s A microbore ODS column (5  was used  for  the LCMS a n a l y s i s  wave l e n g t h U . V . d e t e c t o r  set  of  tocainide glucuronides.  mm I . D . )  A variable  a t 254 nm was placed between the end of  the column and the LCMS i n t e r f a c e glucuronides  u h y p e r s i l ODS, 10 cm x 2.1  jet  to o b t a i n a UV trace of  p r i o r to t h e i r i n t r o d u c t i o n i n t o  The  mobile phase used was a c e t o n i t r i l e : w a t e r  0.2  mL/min and the i n j e c t i o n volume was 2.0  the mass (5:95)  uL.  the  spectrometer.  at a flow r a t e of  For the LCMS s t u d i e s ,  a  Hewlett Packard LCMS system (HP 1090 L i q u i d Chromatograph, 5987A LC -  GCMS system) was used.  The mass spectrometer  i o n i s a t i o n mode w i t h a source  for  tocainide  Both p o s i t i v e  ion  negative  5.  I d e n t i f i c a t i o n o f Band 1^ as T o c a i n i d e Carbamoyl-O-B-D-Glucuronlde  Liquid  were obtained  of 2 0 0 ° C .  i n the chemical  and  5.1.  ion spectra  temperature  was used  glucuronides.  Chromatographic A n a l y s i s o f t h e Hydantoin D e r i v e d from t h e  Glucuronide Tocainide glucuronide, corresponding v a l u e of 0.69 in  water  (isolated  from reverse-phase TLC p l a t e s ) ,  and the pH was adjusted  hydroxide s o l u t i o n .  to band 1^ w i t h an Rf  has  then e x t r a c t e d  dichloromethane.  This  (1980b)  i n formation of a hydantoin-based  to r e s u l t  dissolved  to 13 by the a d d i t i o n of 1.0  The mixture was treatment  was  N sodium  w i t h 5 mL of  been r e p o r t e d by E l v i n e t structure,  al.,  -  3-(2,6-xylyl)-5-methylhydantoin. concentrated residue  was d i s s o l v e d  potassium ODS  on a water  chlorate  column (25  potassium  derivative, liquid  5.2  as  mm I . D . )  to chromatographic  A standard  by A s t r a P h a r m a c e u t i c a l s  i n 0.05 M  i n 0.05 M  hydantoin  was a l s o s u b j e c t e d  a n a l y s i s under i d e n t i c a l c o n d i t i o n s  to  to  compare  times.  GC and GCMS A n a l y s i s o f the H y d a n t o i n D e r i v e d from the G l u c u r o n i d e The  analysed  hydantoin d e r i v a t i v e  by gas  obtained  as d e s c r i b e d  chromatography employing SE-30  column (15 m x 0.2  mm) w i t h flame  chromatograhic retention  identification obtain as  fused-silica  of  The same c a p i l l a r y the h y d a n t o i n .  Methane was used  the chemical i o n i s a t i o n mass s p e c t r a of  as  standard  for  to the the GCMS  the reagent gas  the standard  the h y d a n t o i n d e r i v e d from the g l u c u r o n i d e of  described.  The  to compare  column was a l s o used  also  capillary  was a l s o s u b j e c t e d  a n a l y s i s under i d e n t i c a l c o n d i t i o n s  times.  above was  ionisation detection.  h y d a n t o i n s u p p l i e d by A s t r a P h a r m a c e u t i c a l s  well  The  analysis using a 5 u  and 25% a c e t o n i t r i l e  the mobile phase.  chromatographic  the r e t e n t i o n  bath at 5 5 ° under a stream of n i t r o g e n .  and s u b j e c t e d  supplied  The dichloromethane e x t r a c t was  i n a mixture of 25% a c e t o n i t r i l e  cm x 4.6  chlorate  69 -  to  h y d a n t o i n as  tocainide  as  -  5.3  70  -  A c i d H y d r o l y s i s o f the G l u c u r o n i d e The  Band i s o l a t e d  in  1 mL of 1.0  The  tube was  period,  from the TLC p l a t e ( R f = 0.69)  N HC1 i n a 10 mL screw-capped  t i g h t l y capped and heated at  solution.  (PTFE l i n e d )  dissolved  culture  1 0 0 ° C f o r one hour a f t e r  solvent,  by the a d d i t i o n of 1.0  heptafluorobutyric at 5 5 ° C  i n 200  anhydride were added.  f o r 30 m i n u t e s .  After  The t i g h t l y capped  derivatives  formed by a c i d h y d r o l y s i s of  on a C h i r a s i l - V a l ®  an e l e c t r o n capture  uL of n-hexane.  fused-silica  material  and  the  The h e p t a f l u o r o b u t y r y l the g l u c u r o n i d e  c a p i l l a r y column employing  detector.  Enzyme H y d r o l y s i s o f the G l u c u r o n i d e The  uL of  tube was  room temperature  i n 200  were analysed  the  Excess reagent was removed by  r e s i d u e was r e c o n s t i t u t e d tocainide,  contents  e v a p o r a t i o n of  uL of n-hexane and 30  e v a p o r a t i o n w i t h a slow stream of n i t r o g e n at  of  which  N sodium h y d r o x i d e  i n t o 5 mL of d i c h l o r o m e t h a n e .  the r e s i d u e was d i s s o l v e d  tube.  The excess  An excess of sodium hydroxide was then added and the  were e x t r a c t e d  5.4  was  the r e a c t i o n mixture was c o o l e d to room temperature.  a c i d was n e u t r a l i s e d  heated  t o T o c a i n i d e Enantiomers  (Rf = 0.69)  isolated  dissolved  i n 1 mL of a c e t a t e b u f f e r  added 0.5  mL of  P-glucuronidase  were incubated at 3 7 ° C  t o T o c a i n i d e Enantiomers by p r e p a r a t i v e  (pH = 5.2)  and to t h i s mixture was  enzyme (5000 u n i t s / m L ) .  i n a waterbath  T L C , was  f o r 18 h o u r s .  The c o n t e n t s  The h y d r o l y s a t e was  made a l k a l i n e by a d d i t i o n of 1.0 N sodium hydroxide s o l u t i o n and the contents  of  the tube were e x t r a c t e d  i n t o 5 mL of d i c h l o r o m e t h a n e .  The  -  dichloromethane e x t r a c t chromatographic  6.  71  was processed  -  as d e s c r i b e d  before  for  gas  analysis.  Gas Chromatography/Mass Spectrometry (GCMS) o f T o c a i n i d e Glucuronide s  6.1  Permethylation  of Glucuronides  T o c a i n i d e g l u c u r o n i d e s were permethylated w i t h m e t h y l i o d i d e i n dry  dimethylsulfoxide  c a t a l y s t as procedure,  6.1.1  per  (DMSO) u s i n g m e t h y l s u l f i n y l m e t h i d e c a r b a n i o n  the procedure d e s c r i b e d  described  b r i e f l y , was as  (1973).  The  follows:  P r e p a r a t i o n o f D r y DMSO DMSO was d i s t i l l e d  (20 mL) was added and  by Thompson et a l . ,  as  to 2.0  f r e s h before  the m e t h y l a t i o n p r o c e d u r e .  g of c a l c i u m h y d r i d e i n a round bottom  d i s t i l l e d under vacuum at 7 0 ° C u n t i l  DMSO flask  a 15 mL volume of dry DMSO was  collected.  6.1.2  P r e p a r a t i o n o f Sodium M e t h y l s u l f i n y l m e t h i d e  Carbanion  (Dimsylsodium) A 50% d i s p e r s i o n of sodium h y d r i d e i n m i n e r a l o i l (250 mg, 5 m . moles) quickly DMSO.  was washed  three  transferred  times w i t h anhydrous ether  to a round bottom f l a s k  (3 mL) and was  c o n t a i n i n g 5 mL of dry  The grey c o l o u r e d suspension was heated g e n t l y under n i t r o g e n  -  until  -  f o r m a t i o n of hydrogen ceased ( » 30 m i n u t e s ) .  straw-coloured required  6.1.3  for  s o l u t i o n was  the  stored  under n i t r o g e n a t - 2 0 ° C  sample i s o l a t e d  i n a 5 mL screw capped v i a l  DMSO sodium c a r b a n i o n .  and a f t e r  standing  for  The v i a l  15 minutes  (Reacti-vials®)  was connected  at  hour at  after  room temperature,  a d d i t i o n of 1 mL of d i s t i l l e d  to a n i t r o g e n  which p e r i o d , water.  water.  to a c l e a n v i a l  i n the r e f r i g e r a t o r The  until  same procedure was  followed  freeze-dried  methanol e x t r a c t of  glucuronide,  a pure,  D-glucuronic  acid.  for  oven was  chromatograph was  operated  temperature programmed  stopped  glucuronide layer  times w i t h 1 mL of under n i t r o g e n  and  a n a l y s i s by GC and GCMS.  the p e r m e t h y l a t i o n  commercially a v a i l a b l e ,  of  glucuronide  as w e l l  as  chromatograph was used w i t h a model  5987 Hewlett Packard mass spectrometer/HP gas  three  one  the u r i n e and p - N i t r o p h e n o l  A H e w l e t t - P a c k a r d 5880 gas  The  for  for  The c h l o r o f o r m  then c o n c e n t r a t e d  required  source  uL of  to proceed  The permethylated  and was washed  The c h l o r o f o r m e x t r a c t was  1.2  the r e a c t i o n was  was e x t r a c t e d by shaking w i t h 1 mL of c h l o r o f o r m . transferred  were added 20 uL  room temperature,  The r e a c t i o n was allowed  stored  until  by p r e p a r a t i v e TLC and  m e t h y l i o d i d e were added.  was  resulting  permethylation.  the g l u c u r o n i d e  contained  by  The  Permethylation To  of  72  1000 mini-computer  i n the s p l i t l e s s  system.  i n j e c t i o n mode.  from 5 0 ° to 1 4 0 ° at 3 0 ° C / m i n  The  and from  -  140° 0.2  to 2 4 0 °  at  10°/min.  73  -  An SE-30 f u s e d - s i l i c a  mm) and a C h i r a s i l - V a l ®  mm) were used f o r a n a l y s i s .  fused-silica  c a p i l l a r y column (50 m x  Methane was used as  o p e r a t i o n i n the chemical i o n i s a t i o n mode. used  i n both e l e c t r o n impact ( E I )  a m u l t i p l i e r voltage source  temperature  of  the reagent  gas  The mass spectrometer  and chemical i o n i s a t i o n (CI)  of 2200 v o l t s , 200°.  c a p i l l a r y column (25 m x  e m i s s i o n c u r r e n t of 300  0.31 for was  mode w i t h  pA and a  -  74 -  RESULTS AND DISCUSSION  A.  Gas-liquid  Chromatographic A n a l y s i s  o f Tocainide Using  Fused-silica  C a p i l l a r y Columns The  c o m p l e x i t y of many b i o l o g i c a l samples r e q u i r e s  methods based on h i g h - r e s o l u t i o n chromatography. concerned p r i m a r i l y w i t h the s i t u a t i o n s  analytical  T h i s requirement  is  where multicomponent a n a l y s e s  are needed such as m e t a b o l i c p r o f i l i n g or s c r e e n i n g for numerous drug metabolites,  but i t i s  determinations  also a v a l i d consideration in  of s e l e c t e d  components i n the presence  u n i n t e r e s t i n g compounds or contaminants. the r e s u l t i n g r e s o l u t i o n are popularity  detection is The  attributes  t h i s methodology.  the  increasing  techniques,  r a t i o over  the d e t e c t o r  a s p e c t s of drug  used.  u s i n g a p p r o p r i a t e make-up gas flow i s  The n o i s e  at  of  analysis.  i n an i n c r e a s e d  l e v e l for a c a p i l l a r y  the a p p r o p r i a t e  independent of constant.  is  system  o p t i m i s e d by  flow r a t e .  This  the column flow and the d e t e c t o r  signal-to-noise  is  reduced  they are c o n s t a n t l y purged.  because  are  t y p i c a l peaks obtained w i t h packed columns,  g e n e r a l l y reduced because the d e t e c t o r performance  make-up gas  there  High s e n s i t i v i t y  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  r e g a r d l e s s of  Contaminants from the septum are  to the s m a l l amount of the s t a t i o n a r y is  for  chromatographic  of c r u c i a l importance i n v a r i o u s  signal-to-noise  is  of  of m e t a b o l i c a l l y  While column e f f i c i e n c y and  the c h i e f reasons  of c a p i l l a r y column gas  other v a l u a b l e  trace  more uniform because f l u c t u a t i o n s  Column bleed i s  phase present  also  reduced due  and c a r r i e r gas  flow  are dampened by the r e s i s t a n c e of  -  the column. decreased, ratio.  75 -  C o n s e q u e n t l y , peak h e i g h t i s often r e s u l t i n g  This increased  g a i n i n the  s e n s i t i v i t y extends  the chromatographic system. certain applications.  i n a 100:1  increased  is  signal-to-noise  the dynamic l i n e a r range of  The s e n s i t i v i t y  Another reason f o r  and the n o i s e  g a i n may be c r u c i a l i n  the i n c r e a s e d  sensitivity  i n h e r e n t w i t h c a p i l l a r y columns i s  due  stationary  The degree of sample a d s o r p t i o n and  phase and i t s  support.  to the i n e r t nature  d e c o m p o s i t i o n o c c u r r i n g on the column i s labile  substances of p h a r m a c e u t i c a l  especially  true of f u s e d - s i l i c a  from f u s e d - s i l i c a ,  which i s  enough to be n e a r l y unbreakable  of  such columns, are  mixture". groups.  chromatographic  the f u s e d - s i l i c a  columns and the  assessed by the a n a l y s i s  u t i l i t y for a  problem can be determined by c a r e f u l l y  p o l a r i t y mixture.  fused-silica  carbowax 20 M.  but  "polarity functional specific  examining peak  shapes  F i g u r e 2 shows a  The column used was a 50 m x 0.2 mm.  column that had been coated with a 2% s o l u t i o n of  The chromatogram was obtained on a model 5830 A gas  chromatograph (Hewlett-Packard)  equipped with s p l i t  injection  i o n i s a t i o n and e l e c t r o n  system and both flame  are  efficiency  of a so c a l l e d ,  the p o l a r compounds c o n t a i n e d i n t h i s m i x t u r e .  I.D.  performance  i n normal usage.  The column's s u r f a c e a c t i v i t y and i t s  chromatogram of  is  1 ppm m e t a l l i c  T h i s mixture i n c l u d e s compounds w i t h a v a r i e t y of  separation of  of  This  Columns prepared  pure s i l i c o n d i o x i d e (<  flexible  The i n e r t nature  can be a n a l y s e d .  c a p i l l a r y columns.  p r o v i d e not only e x c e l l e n t  the  minimized and t h e r e f o r e many  interest  oxides),  of  and  splitless capture  - 76 -  FIGURE 2 CHROMATOGRAM ON  CARBOWAX  20M  OF POLARITY  FUSED  MIXTURE  SILICA C A P I L L A R Y  1. 2. 3A. 56. 7. 8. 9.  10  20  COLUMN  NONANE DECANE UNDECANE DODECANE DIBUTYLKETONE TETRADECANE CIS-PROPYLCYCLOHEXANOL TRANS-PROPYLCYCLOHEXANOL 2,6-DIMETHYLAN I LINE  30  MINUTES Chromatographic C o n d i t i o n s : Column, Carbowax 20 M (50 m x 0.2 mm); I n j e c t i o n temperature, 260°C; Detector (F.I.D.) temperature, 260°C; Column temperature, 70°C (5 min) to 220°C at a rate o f 10°C/min; C a r r i e r gas (Helium) flow, 1 ml/min; S p l i t vent f l o w , 130 ml/min; I n l e t p r e s s u r e , 25 p.s.i ; Make-up gas (Helium) flow, 50 ml/min; Chart speed, 0.3 cm/min.  -  detectors.  77  -  The flame i o n i s a t i o n d e t e c t o r  was used i n t h i s case and the  sample was i n t r o d u c e d by the s p l i t mode of  injection.  No t a i l i n g was observed f o r the k e t o n e , a l c o h o l or the amino compounds contained for  i n the p o l a r i t y m i x t u r e .  the two i s o m e r i c a l c o h o l s ,  Base-line  r e s o l u t i o n was o b t a i n e d  showing the e f f i c i e n c y as w e l l as  the  i n e r t n a t u r e of the column. T o c a i n i d e was f i r s t form a d e r i v a t i v e .  reacted  w i t h h e p t a f l u o r o b u t y r i c anhydride to  The chromatogram obtained by i n j e c t i n g a s o l u t i o n o f  the h e p t a f l u o r o b u t y r a t e  of t o c a i n i d e i n n-hexane at a s p l i t  1:50  3.  is  shown i n f i g u r e  w i t h argon-methane  A  (95:5) as  . ^ ^ i electron-capture  the make-up g a s .  peak o b t a i n e d was c o n s i d e r e d s u p e r i o r as o b t a i n e d on S i l a r (figure  4,  derivative  10 C , OV-225 or SP-2330  a , b and c ) . (7.6  m i n . at  considered suitable where a l a r g e The  detector  was  of used  The sharp symmetrical  compared to  chromatograms  g l a s s c a p i l l a r y columns  The r e t e n t i o n time of the peak of a column temperature  for a n a l y s i s  ratio  of 2 0 0 ° C )  was  tocainide also  of t o c a i n i d e i n a b i o l o g i c a l m a t r i x  number of peaks u s u a l l y appear  same carbowax 20 M f u s e d - s i l i c a  near the s o l v e n t  c a p i l l a r y column was used  front. to  o p t i m i z e c o n d i t i o n s of a c y l a t i o n of t o c a i n i d e with h e p t a f l u o r o b u t y r i c anhydride. this  a-Bromonaphthalene was chosen as  study because i t  is  unaffected  electron-capturing properties.  an i n t e r n a l standard  by the presence  A p l o t of  of HFBA and has good  the area r a t i o s  measured  different  time p e r i o d s of h e a t i n g the r e a c t i o n mixture at 5 5 ° C i s  in  5.  figure  There was a sharp d e c l i n e i n the area r a t i o s  samples which had been heated  in  f o r 75 minutes or more.  at shown  measured  However, the  for  -  78 -  FIGURE 3 CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVE OF TOCAINIDE ON CARBOWAX 20 M FUSED-SILICA CAPILLARY COLUMN  Chromatographic c o n d i t i o n s : 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). I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) t e m p e r a t u r e , 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas (argon:methane ( 9 5 : 5 ) f l o w , 60 mL/min; column i n l e t p r e s s u r e , 172.3 k P a ; c h a r t speed, 0.3 cm/min; s p l i t vent f l o w , 50 mL/min.  -  79  -  FIGURE 4 CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVE OF TOCAINIDE  Chromatographic A.  B.  C.  conditions:  Column, s i l a r 10 C g l a s s 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 ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature; 2 3 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas (argon:methane (95:5) f l o w , 60 mL/min; column i n l e t p r e s s u r e , 34.4 kPa; c h a r t speed, 0.3 cm/min; s p l i t vent f l o w , 40 mL/min. Column, 0V-225 g l a s s 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 p r e s s u r e 137 k P a . Other c o n d i t i o n s are the same as in A. Column, SP-2330 g l a s s 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 p r e s s u r e , 103.4 kPa. Other c o n d i t i o n s are the same as i n A .  - 80 -  FIGURE 5 TIME-DEPENDENCE OF DERIVATIVE FORMATION  -  area  ratios  were n e a r l y constant  to 60 m i n u t e s . period for  1.  81 -  f o r a l l samples heated  T h e r e f o r e , 30 minutes was chosen as a reasonable  a-Bromonaphthalene was used as e a r l y development of  it  Although t h i s  because of I t s 6),  an i n t e r n a l standard  as  an i n t e r n a l standard  very low r e t e n t i o n t i m e .  a metabolite  properties,  i n the present  M o n o e t h y l g l y c i n e x y l i d i d e (MEGX)  t o c a i n i d e and has  a reasonably  the e a r l y e l u t i n g endogenous  long compounds  MEGX-HFB a l s o has good chromatographic  gave a symmetrical peak on the carbowax  CH  study  of l i d o c a i n e , does form a d e r i v a t i v e with HFBA  r e t e n t i o n time and t h e r e f o r e , would not i n t e r f e r e .  d u r i n g the  plasma and  compound had good e l e c t r o n c a p t u r i n g  under the same c o n d i t i o n s as  and  Standard  t o c a i n i d e i n an ECD assay f o r r a t  was not found s u i t a b l e  (figure  time  the d u r a t i o n of d e r i v a t i z a t i o n .  Selection of a Suitable Internal  urine.  from 15 minutes  column.  3  H NH—CO—CH —N 2  C H 2  FIGURE 6  5  properties  - 82  2.  Measurement o f S p l i t t e r D i f f e r e n t i a t i o n Between T o c a i n i d e and Internal  S t a n d a r d , MEGX  A capillary inlet splitter portions,  the s m a l l e r  'splits'  the sample i n t o two unequal  of which goes onto the column.  T h i s reduces  amount of the sample going through the column and prevents loading.  The major  restrict  f u n c t i o n of the i n l e t s p l i t t e r  column o v e r -  not o n l y  to  the s i z e of the sample placed on the column but a l s o to permit  the r a p i d f l u s h i n g of the i n j e c t i o n port column i s diluted  is  the  f o l l o w e d by pure c a r r i e r gas  sample.  Split  injection is  so that  rather  the sample on the  than by e x p o n e n t i a l l y  a flash vaporisation  technique,  and  t h e r e e x 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 .  Discrimination is  a measure of how w e l l  the o r i g i n a l  sample c o m p o s i t i o n .  the d e t e c t e d Differences  peak areas r e f l e c t  i n molecular weight,  component c o n c e n t r a t i o n , p o l a r i t y , temperature  split  and p r e s s u r e may a l l a f f e c t  T h e r e f o r e i t was n e c e s s a r y to t e s t t i o n of  for  t o c a i n i d e and i n t e r n a l standard  keeping a l l o t h e r c o n d i t i o n s Split  r a t i o s used f o r  i n j e c t i o n volume (1 were recorded ( T a b l e significant  this  c o n d i t i o n s of the a n a l y s i s  the nature of the the p o s s i b i l i t y of under d i f f e r e n t  study ranged from 1:15 all  These r e s u l t s  differentiation  i n j e c t e d volume,  inlet  split. differentia-  split  ratios,  constant.  uL) was used at 1).  ratios,  volatility,  split  to 1:130.  r a t i o s and the area  showed that  Same ratios  there was no  between t o c a i n i d e HFB and MEGX HFB under since  the area r a t i o s were n e a r l y  the  constant.  Table 1 Area r a t i o s  split  at d i f f e r e n t  ratio  split  area  130 100 75 50 25 15  A single data  3.  split  ratio,  ratios  ratio  1.345 1.376 1.286 1.363 1.308 1.357  o p t i m i s e d f o r the d e t e r m i n a t i o n of  and p r e c i s i o n were used f o r the assay of b i o l o g i c a l  calibration samples.  Minimum D e t e c t a b l e Q u a n t i t y In  o r d e r to determine the lower l i m i t  determination  of t o c a i n i d e ,  of s e n s i t i v i t y f o r  picogram q u a n t i t i e s  were i n j e c t e d onto  carbowax 20 M c a p i l l a r y column and the s i g n a l - t o - n o i s e observed.  F i g u r e 7 shows the d e t e c t o r  t o c a i n i d e a r r i v i n g at  the d e t e c t o r  ratio  r a t i o of 1:30  The  volume i n j e c t e d was 1 pL which c o n t a i n e d 90 picograms of  4.  Application  the  was  response to 3 picograms  when a s p l i t  the  of was  used.  tocainide.  o f C a p i l l a r y gas chromatography f o r t h e A s s a y o f Rat  Plasma and U r i n e In  order to demonstrate  c a p i l l a r y column gas check the r e s u l t s containing  the a p p l i c a b i l i t y of the newly developed  chromatographic assay to b i o l o g i c a l samples and to  with published data,  t o c a i n i d e were analysed  rat  plasma and u r i n e samples  ( F i g u r e 8, A and B ) .  F i g u r e 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 c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; column i n l e t p r e s s u r e , 172.3 k P a , c a r r i e r gas (He) flow r a 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; c h a r t 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. M E G X  X^jtrtank plasma  Chromatographic  V  blank urine  conditions:  A.  I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven t e m p e r a t u r e , 2 0 0 ° C ; c a r r i e r gas (He) flow r a t e , 1 mL/min; make-up gas (argon:methane, 9 5 : 5 ) , 60 mL/min; column i n l e t p r e s s u r e , 172.3 kPa; s p l i t vent f l o w , 30 mL/min; c h a r t speed, 0.3 cm/min.  B.  Oven temperature,  195°C.  A l l other  conditions  are  the same as  in A.  - 86 -  the r a t  plasma  p r o f i l e 3 hours a f t e r  an i n t r a v e n o u s  dose of  h y d r o c h l o r i d e along with the ECD t r a c e of blank plasma. peaks were observed well resolved  No i n t e r f e r i n g  i n the plasma and the i n t e r n a l standard  from t o c a i n i d e .  In the r a t  tocainide  u r i n e ( F i g u r e 8B)  (MEGX) was profile  t h e r e was a s m a l l peak e l u t i n g v e r y c l o s e to t o c a i n i d e which was r e s o l v e d by r e d u c i n g the temperature  of a n a l y s i s  to  195°C.  C a l i b r a t i o n d a t a and p r e c i s i o n of assay f o r r a t are of  shown i n Table 2. each of s i x  along with 1000 ng of  the i n t e r n a l s t a n d a r d .  The mean r e l a t i v e  the plasma and u r i n e ,  standard  respectively.  P h a r m a c o k i n e t i c s o f I n t r a v e n o u s T o c a i n i d e i n t h e Rat Rat plasma  and u r i n e were analysed  chromatographic method.  by the c a p i l l a r y column gas  Plasma d a t a were analysed  (AUTOAN and NONLIN) and were found to f i t  levels  of t o c a i n i d e are shown i n Table 3,  parent  drug e x c r e t e d  Table  program  three d i f f e r e n t  The dose  and the cumulative amount of  i n the u r i n e up to a p e r i o d of 24 hours i s  shown i n  4. As  shown i n Table 3, w i t h i n c r e a s i n g d o s e s ,  plasma c o n c e n t r a t i o n - t i m e curve i n c r e a s e d decreased.  C l e a r a n c e should be c o n s t a n t  observation  is  as  by a computer  a two compartment model.  pharmocokinetic parameters i n male W i s t a r r a t s at  rat  analyses  of t o c a i n i d e ranging from 50 to 1000 ng  d e v i a t i o n was 8.3% and 6.1% f o r  5.  plasma and u r i n e  These data were determined by t r i p l i c a t e  concentrations  well  i n agreement  reported e a r l i e r  observations  the area under  and the t o t a l if  clearance  linear kinetics apply.  with pharmacokinetics  of t o c a i n i d e  (Venkataramanan and A x e l s o n , 1980).  also indicate  that  the  This  i n the  These  a simple l i n e a r pharmacokinetic model  is  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 f o r r a t plasma and u r i n e assay  w t . of t o c a i n i d e (ng)  plasma area r a t i o + S.D.  u r i n e area ratio + 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  Correlation  coefficient, r =  0.9989  0.9989  slope  =  1.3058  1.3456  intercept  = -0.0168  -0.0209  0.1013  -'88 -  Table 3 P h a r m a c o k i n e t i c parameters of i n t r a v e n o u s  tocainide  dose :25 mg/kg Rat #1 K ' el  (min-1)  Rat #2  i n the r a t s  20 mg/kg Rat #i3  15 mg/kg  Rat #4.  Rat M§  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 (rain-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  K12  (min-1)  0.0048  0.0249  0.0692  0.0983  0.0221  K21  (min-1)  0.0079  0.0489  0.0512  0.0484  0.0325  T1/2(g)(min) AUC ( y g . h m L - 1 ) CI (mL/min)  V d (mL)  183.0  152  143.0  22.58  23.54  22.11  3.98  3.85  3.77  1049.0  858.0  785.0  163.0  142.0  16.6  10.4  4.45 1061.0  9.10 1854.0  -  89 -  Table 4 P e r c e n t drug e x c r e t e d unchanged i n 24 h u r i n e  Dose 10 mg/kg Rat  Dose 20 mg/kg  #1  Rat  14.2  inadequate present  #2  Rat  21.6  to d e s c r i b e  #3  29.8  the k i n e t i c s of  t o c a i n i d e i n the r a t .  In the  case o n l y 3 dose l e v e l s were s t u d i e d i n a l i m i t e d number of  rats.  B.  R e s o l u t i o n o f T o c a i n i d e Enantiomers T o c a i n i d e has one c h i r a l  stereoisomeric  forms  (figure  c e n t e r and t h e r e f o r e  9).  It  is  can e x i s t  used c l i n i c a l l y  which c o n t a i n s equal p r o p o r t i o n s of both the i s o m e r s . that  the l e v o r o t a t o r y isomer i s  about  1979).  Differences  i n a b i o l o g i c a l system.  i n t e r a c t i o n would be the r e c e p t o r the r e c e p t o r 10).  s i t e i t undergoes  i n a mouse model (Byrnes  site itself,  stereoselective  T h u s , a b s o r p t i o n from the g a s t r o i n t e s t i n a l  et  al.,  1975;  Jenner et a l . ,  enantiomers  area f o r such an  but before  v a r i o u s body compartments and m e t a b o l i c pathways (Patil  et  i n t e r a c t w i t h asymmetric  The most l i k e l y  several  racemate  has been shown  i n p h a r m a c o l o g i c a l a c t i v i t y between  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 molecules  It  the  3 - f o l d more potent as an  a n t i a r r h y t h m i c agent than the dextroisomer al.,  as  i n two  1980;  events  reaches  (Figure  tract,  d i s t r i b u t i o n to  can be  stereoselective  Low et a l . ,  amount of each isomer r e a c h i n g the r e c e p t o r s i t e ,  a drug  1978).  The f i n a l  therefore,  depends on  - 90 -  FIGURE 9 STRUCTURES OF TOCAINIDE ENANTIOMERS  R(—)TOCAINIDE  H  H  ,  C  H . C < M - 0 °  H3C  S(+)TOCAINI  DE  -  91  -  FIGURE 10 STEREOSELECTIVE EVENTS PRIOR TO BIOLOGICAL RESPONSE  DRUG DOSE  MEMBRANE SELECTIVITY  BIOLOGICAL RESPONSE  SELECTIVE METABOLISM  DRUG RECEPTOR  -  the degree of s t e r e o s e l e c t i v i t y s t u d i e s of enantiomers the a b s o r p t i o n  that  is  capable  In order to study the d i s p o s i t i o n of and s e l e c t i v e  analytical  of r e s o l v i n g the two isomers  fluids.  r e s o l u t i o n of  Pharmacokinetic  can r e v e a l whether the s t e r e o s e l e c t i v i t y  a very s e n s i t i v e  biological  of these p r o c e s s e s .  lies  technique i s  when present  Such a method has been r e p o r t e d  t o c a i n i d e enantiomers  enantiomeric  for  required  together  the  (McErlane and P i l l a i ,  in  direct  1983).  This  method i n v o l v e s the r e s o l u t i o n of the h e p t a f l u o r o b u t y r y l d e r i v a t i v e s t o c a i n i d e on a c a p i l l a r y column coated w i t h an o p t i c a l l y stationary  phase, N - i s o b u t y r y l - L - v a l i n e t e r t - b u t y l a m i d e  C a p i l l a r y columns coated w i t h t h i s available ed  al.,  Solomon and W r i g h t ,  to employ a mass spectrometer enantiomers  ( L i u et a l . ,  Frank et  (Figure  11).  alcohols  In a d d i t i o n , the h i g h  possible,  al.,  demonstrat-  and amino  for  coupled to a GLC system  1981:  active  and have been  1977).  of C h i r a s i l - V a l ® made i t  of  phase are c o m m e r c i a l l y  f o r the r e s o l u t i o n of amino a c i d s  1980;  thermal s t a b i l i t y  of  stationary  under the trade name, C h i r a s i l - V a l ® ,  to be e f f e c t i v e  (Frank et  in  p r o c e s s , d i s t r i b u t i o n , metabolism or a combination of  these p r o c e s s e s . drugs,  92 -  the f i r s t  for  the  time,  analysis  1978).  F i g u r e 12 shows the chromatogram of the h e p t a f l u o r o b u t y r y l derivative extracted  of t o c a i n i d e enantiomers  from a 24 hour u r i n e sample from a r a t  tocainide hydrochloride. No.  2)  and R ( - ) t o c a i n i d e  chromatographic derivative  and of the i n t e r n a l  Base-line  standard  dosed w i t h ( ± )  r e s o l u t i o n of S ( + ) t o c a i n i d e  (peak No. 3) was o b t a i n e d w i t h a  time of l e s s  than 9 m i n u t e s .  (peak  total  The h e p t a f l u o r o b u t y r y l  of m o n o e t h y l g l y c i n e x y l i d i d e ( i n t e r n a l standard) had a r e t e n -  - 93 -  FIGURE 11  STRUCTURE  O F T H E CHIRAL STATIONARY  chirasil-val  PHASE  - ;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 c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 8 0 ° C ; c a r r i e r gas (He) flow r a t e , 1 mL/min; s p l i t vent f l o w , 25 mL/min; make-up gas (argon:methane, 9 5 : 5 ) , 60 mL/min; column i n l e t p r e s s u r e , 103.4 kPa; c h a r t speed, 0.3 cm/min.  -  tion  time s h o r t e r  95 -  than that of t o c a i n i d e on the c h i r a l  c a p i l l a r y column  ( u n l i k e t h e i r e l u t i o n p a t t e r n from a carbowax 20 M c a p i l l a r y column where r e t e n t i o n time of MEGX-HFB was l o n g e r than t o c a i n i d e H F B ) . 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  tocainide  mers on a c a p i l l a r y column coated with o p t i c a l l y a c t i v e phase.  i n the r a t  i n peak h e i g h t of the enantiomers. when the racemic drug was analysed  1.  stationary  as e v i d e n t from the  Equal peak h e i g h t s (Figure  disposidifferences  were o b t a i n e d  13).  I d e n t i f i c a t i o n o f t h e R e s o l v e d Peaks The  synthesis  i d e n t i t y of the peaks was e s t a b l i s h e d of the pure isomers  heptafluorobutyrates  from a  using s t a r t i n g materials  p u r i t y and c o n f i g u r a t i o n ( F i g u r e  and  enantio-  T h i s chromatogram a l s o c l e a r l y showed the d i f f e r e n t i a l  t i o n of t o c a i n i d e enantiomers  This  14).  stereospecific of known o p t i c a l  The r e t e n t i o n time of  of the s y n t h e t i c p r o d u c t s ,  their optical  the mass s p e c t r a were used f o r i d e n t i f i c a t i o n ( F i g u r e s  Furthermore,  the rotation  15,  16).  the o p t i c a l p u r i t y was determined by measuring the  area  under the two peaks. Stereospecific  synthesis  the method of Byrnes et a l . ,  of t o c a i n i d e enantiomers was achieved by  (1979).  It  consisted  of f o r m a t i o n o f an  amide bond between an amino a c i d and an aromatic amine and i s to procedures e s t a b l i s h e d  for peptide s y n t h e s i s .  a l a n i n e , a commercially a v a i l a b l e to r e a c t  analogous  Carbobenzyloxy D(-)  amine-protected amino a c i d was allowed  with 2 , 6 - d i m e t h y l a n i l i n e i n the presence of d i c y c l o h e x y l -  carbodiimide.  One of the advantages of carbobenzyloxy amino a c i d s  is  FIGURE 13  CHROMATOGRAM OF  (±) TOCAINIDE H E P T A F L U O R O B U T Y R A T E S .  Column: C h i r a s i l - v a l g l a s s capillary column (25Mx0.25mm)  S(+)tocainide 7.9mins R ( - ) t o c a i n i d e 8.4mins  Chromatographic c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature, 1 8 0 ° C ; c a r r i e r gas (He) flow r a t e , 1 mL/min; s p l i t vent f l o w , 25 mL/min; make-up gas (argon:methane, 9 5 : 5 ) , f l o w , 60 mL/min; chart speed, 0.3 cm/min; column i n l e t p r e s s u r e , 103.4 k P a .  -  97 '--  FIGURE  14  SYNTHESIS OF R(-) TOCAINIDE .CH C H j C H COOH NH C O O C H C H 2  s  3  if*  +  (\  5  /V-NH CH  carbobenzyloxy D-alanine  3  2  2,6-dimethyl aniline  dicyclohexylcarbodiimide (DCC) CH  V  3  y—NHCOCHNH \  CH,  COOCH C H 2  CH,  HBr/HAc  U  A—NHCOCHNH 2  \_  HBr  CH,  R(-) Tocainide hydrobromide  6  5  -  t h e i r general employed f o r protective  r e s i s t a n c e to r a c e m i z a t i o n under c o n d i t i o n s the formation of  effect  possesses f u r t h e r  1963).  peptide bonds.  against racemization,  such as  usually  In a d d i t i o n to  the carbpbenzyloxy  advantage i n the ease with which i t  by a number of methods temperature  98 -  catalytic  its  group  may b'e-<rembved  hydrogenation at room  or the use of hydrogen bromide i n a c e t i c  acid  (Boissonnas,  The product of the r e a c t i o n between carbobenzyloxy D(-)  and 2 , 6 - d i m e t h y l a n i l i n e , on treatment bromide i n a c e t i c  acid,  with a 32% s o l u t i o n of hydrogen  gave a p r e c i p i t a t e  hydrobromide upon a d d i t i o n of e t h e r .  c o m p o s i t i o n o f 95:5  of the R(-)  tocainide  Ether also dissolved  benzylbromide formed d u r i n g the r e a c t i o n . enantiomeric  alanine  the  The product obtained  of the R ( - ) : S ( + )  isomers,  as  had an  calculated  from the peak areas when t h e i r h e p t a f l u o r o b u t y r y l d e r i v a t i v e s  were  assayed on the C h i r a s i l - V a l ®  evidence  supported  the s t r u c t u r e  p r o f i l e of  c a p i l l a r y column.  of the major  isomer.  the h e p t a f l u o r o b u t y r y l d e r i v a t i v e  Mass s p e c t r a l The t o t a l  of R(-)  ion current  tocainide  i n F i g u r e 15 and the e l e c t r o n impact mass s p e c t r a i n f i g u r e  2.  is  shown  16.  C a l i b r a t i o n Data and P r e c i s i o n o f A s s a y o f T o c a i n i d e Enantiomers i n Human Plasma and U r i n e 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  in  t a b l e 5,  and the c a l i b r a t i o n d a t a and p r e c i s i o n of assay i n u r i n e  shown i n t a b l e 6.  For the plasma  area r a t i o s were measured eluting  closely  same r e a s o n ,  are shown  to  a s s a y , peak h e i g h t r a t i o s r a t h e r  because o f an i n t e r f e r i n g endogenous  the peaks f o r  the t o c a i n i d e  enantiomers.  the plasma assays had to be c a r r i e d out at a  are  than  compound  For the slightly  --99. FIGURE 15  TOTAL ION CURRENT PROFILE OF HEPTAFLUOROBUTYRYL DERIVATIVE OF R(-) TOCAINIDE  200  468 i  zeeee-  608  880  1B0B  • '• •  i •  l  1288 I:  i•  1400 • ' i. .  I , li  188G9160091400012000108008300-  eaea40002BB0-' e-  •  8  !  • 19  • 12  .  . 14  r— r~—i 16  r  _  i i —r——i  18  28  i •i 22  1 r~—i 1 1 1 i 1  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 ® g l a s s 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 t e m p e r a t u r e , 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 ; a n a l y s e r temperature, 2 4 0 ° C ; t r a n s f e r l i n e t e m p e r a t u r e , 2 4 0 ° C ; i o n i s a t i o n p o t e n t i a l , 70 e v .  -  100  -  FIGURE 16  El MASS SPECTRA OF HEPTAFLUOROBUTYRYL DERIVATIVES OF TOCAINIDE ENANTIOMERS Sc an 4 I B 11.27 < i n  ! i ea  RHTOCAINIDE HFB H,C  H,C'S / -\J> N  14B  C  fjC F,C F,COCH N  i\ O  H,C  M  388  5B  IBB  I5B  286  ^SB  3BB  35B  4SB  ABB  58B  S5B Sc a n 3 7 7 13 . 62 m i n .  S(+)TOCAINIDE HFB  /  CH  241 l  H  CH,  24 1  O  NH COiCFjCFjCF, :OiCFj  _  148  1«»  M  388  169  2<H  IBB  1SB  299  256  388  3SB  488  458  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 enantiomers i n human plasma  tocainide  q u a n t i t y of each enantiomer (ng)  S(+)T0C  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  peak h e i g h t r a t i o s + S. D .  Correlation coefficient,  RSD(%)  R(- )T0C  r =  0.9998  0.9989  slope  =  0.9024  0.8171  intercept  = --0.0012  0.0018  The q u a n t i t y o f i n t e r n a l s t a n d a r d was 1 u g .  RSD(%)  -  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 enantiomers i n human u r i n e  q u a n t i t y of each enantiomer  tocainide  area r a t i o s ± S . D . S(+)T0C  (ug)  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  5.4  1.1590  ± 0.0080  0.7  Correlation coefficient,  ± 0.0570  r =  0.9994  0.9984  slope  =  0.9851  1.0982  intercept  = --0.0596  0.0469  The q u a n t i t y o f i n t e r n a l standard  was 1 ug.  - 103 -  different  column temperature.  the i n t e r f e r i n g peak as errors  as  possible  i n q u a n t i t a t i o n were m i n i m a l .  observed  i n u r i n e or s a l i v a  determine  triplicate  the gas  Inter  samples ( F i g u r e s  17A, B & C ) . under  chromatographic a s s a y ,  sample v a r i a t i o n s  intra-sample v a r i a t i o n s  (Table  to  resolve that  No such i n t e r f e r i n g peaks were In order  to  the optimum  triplicate  samples were made and the r e l a t i v e  calculated.  was necessary  from the t o c a i n i d e peaks so  the r e p r o d u c i b i l i t y of area r a t i o s  c o n d i t i o n s of  3.  far  T h i s adjustment  injections  standard d e v i a t i o n s  of were  were found to be h i g h e r than  7).  P r e l i m i n a r y Study o f S t e r e o s e l e c t i v i t y i n T o c a i n i d e D i s p o s i t i o n i n Man Two h e a l t h y male v o l u n t e e r s  chromatographic p r o f i l e s dose of  measured  in  i n both s u b j e c t s .  2.53  are g i v e n i n T a b l e 8.  and 1.49,  and i n s u b j e c t 2, respectively,  1.49.  after  gas  an o r a l  plasma  of  the  the enantiomer  By 48 hours the  i n the two s u b j e c t s . by a  The  The plasma c o n c e n t r a t i o n  By 24 hours p o s t - d o s i n g  R(-)  ratio  ratios  The plasma  non-stereospecific  chromatographic method employing a carbowax 20 M c a p i l l a r y  column which does n o t r e s o l v e can  study.  u r i n e and s a l i v a  shown i n F i g u r e 17 and the  samples from s u b j e c t 2 were a l s o analysed capillary  i n this  at each time p e r i o d showed more r a p i d disappearance  s u b j e c t 1 was 1.94  were  t h e i r plasma,  the racemic drug are  c o n c e n t r a t i o n - t i m e data  isomer  of  participated  be observed  that  the racemate  the t o t a l  when assayed i n d i v i d u a l l y u s i n g agreement with the q u a n t i t y of  quantities  ( T a b l e 8 and Table 9 ) . of S(+)  the C h i r a s i l - V a l ® the racemate  and R(-)  It  enantiomers  column are  in  close  employing the carbowax  - ioU FIGURE  IT  PLASMA, URINE. AND SALIVA PROFILE 24 HOURS AFTER AN ORAL DOSE OF ( ± ) T O C A I N I D E  HYDROCHLORIDE 1. internal standard 2S(+) Tocainide 3-R(-) Tocainide  1  A  3  JJUL —i— 10  PLASMA  1 0  URINE  10  SALIVA  Chromatographic c o n d i t i o n s : A. Column, C h i r a s i l - V a l * g l a s s c a p i l l a r y column (25 m x 0.25 mm); I n j e c t i o n temperature, 240°C; d e t e c t o r (ECD) temperature, 350°C; oven temperature, 183°C; c a r r i e r gas (He) f l o w , 1 mL/min; s p l i t vent flow, 30 mL/min; make-up gas (argon:methane (95:5) f l o w , 60 mL/min;'column i n l e t p r e s s u r e , 103.4 kPa; c h a r t speed, 0.3 cm/min. B & C. Oven temperature 180°C. A l l other c o n d i t i o n s are same as i n A.  -  105 -  Table 7 I n t e r - and i n t r a - s a m p l e 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. 2. 3. mean ± SD RSD(%)  Sample 2  1. 2. 3. mean ± SD RSD(%>  Sample 3  mean ± SD RSD (%) mean of 3 samples RSD (%)  1. 2. 3.  0.5695 0.5512 0.5866 0.5691 3.0  ± 0.0177  0.6525 0.5762 0.6121 0.6136 ± 0.0381 6.2  0.6503 0.6090 0.6207 0.6266 ± 0.0212 3.3  0.6788 0.6242 0.6503 0.6511 4.1  0.5330 0.5259 0.5206 0.5265 1.1  0.5626 0.5907 0.5444 0.5659 4.1  ± 0.0062  0.5740 ± 0.0402 7.0  ± 0.0273  ± 0.0233  0.6102 ± 6.9%  .0427  -  106 -  Table 8 Plasma c o n c e n t r a t i o n (ug/mL)-time d a t a f o l l o w i n g an o r a l dose of 3 mg/kg t o c a i n i d e  Chiral Subject 1  Time (h)  Carbowax (20M)  Column Subject 2  Subject 2  s(+)  R(-)  s(+)  R(-)  Total  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  0.049  0.032  0.081  0.086  72.00  -  -  -  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 u s i n g carbowax 20 M c a p i l l a r y column  Quantity  of T o c a i n i d e  (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  '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  200.0  Correlation coefficient,  '  r =  0.9994  Slope  =  1.2313  Intercept  =  0.001  The q u a n t i t y of i n t e r n a l s t a n d a r d was 1 u g .  -  column.  The h a l f - l i v e s  the two v o l u n t e e r s  of  108 -  t o c a i n i d e enantiomers  are g i v e n i n T a b l e  and of the racemate  in  10.  T a b l e 10 Half-lives  (h)  of  t o c a i n i d e enantiomers and the racemate healthy volunteers  Subject  1  Subject 2  S(+)  Tocainide  11.1  25.6  R(-)  Tocainide  9.0  20.5  9.2  22.1  R,S(±)  The  Tocainide  Chirasil-Val®  performance d e t e r i o r a t e d extent  of i n t e r f e r e n c e  g l a s s c a p i l l a r y column (25 m x 0.25 mm) w i t h repeated  a n a l y s e s of plasma  from the c l o s e l y e l u t i n g endogenous  more pronounced making q u a n t i t a t i o n u n r e l i a b l e a f t e r further  a n a l y s e s of plasma,  utilizing a fused-silica the same s t a t i o n a r y resulted  Chirasil-Val®.  e l i m i n a t i o n of i n t e r f e r e n c e  The  r e s o l u t i o n of  The use  The  became  Therefore,  of  mm) coated this  the two isomers  with  column but a l s o  the  from the endogenous substance i n plasma  18).  i n t e r n a l standard  used i n t h i s case was  a c e t o x y l i d i d e , W-49167 ( A s t r a P h a r m a c e u t i c a l s ) related  peak  a time.  c a p i l l a r y column (50 m x 0.3  phase,  samples.  u r i n e and s a l i v a were c a r r i e d out by  not only i n b e t t e r  samples ( F i g u r e  i n two  in structure  ( F i g u r e 19)  to t o c a i n i d e  1-amino  which i s more c l o s e l y than monoethyl g l y c i n e  -  109. -  FIGURE 18  TOCAINIDE P L A S M A WITH  ENANTIOMERS  0.2JUG O F  E A C H ENANTIOMER  A N D 1JJG O F I N T E R N A L 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.  0  10  20  internal standard  30  MINUTES  Chromatographic c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) t e m p e r a t u r e , 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas(N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; c h a r t speed, 0.3 cm/min.  FIGURE- 19 STRUCTURES OF 1-AMINOACETOXYLIDIDE (W-49167) AND TOCAINIDE  NHCOCHNHo  I  CHj  Tocainide  CH,  ^_V-NHCOCH NH a  z  CH,  Internal  standard  -  xylidide.  Ill  A primary amine l i k e  t o c a i n i d e , W-49167 a l s o r e a c t s  produce a monoheptafluorobutyrate tocainide derivatisation additional  advantage  derivative  (Figure 20).  that i t  metabolites  such a s i t u a t i o n  it  further  internal  standard.  an  dose of  oral  92 hours a f t e r of S(+)  indicative  4.  to assay plasma  present  a metabolite  has  the  samples even  (MEGX cannot be used i n  of l i d o c a i n e ) .  Therefore,  all  the i n t e r n a l  T a b l e 11 and t a b l e 12 show the c a l i b r a t i o n d a t a and p r e c i s i o n  assay f o r human plasma  heights  are  to  the c o n d i t i o n s of  assays were c a r r i e d out by u s i n g W-49167 as  standard. of  is  under  T h i s i n t e r n a l standard  can be used  when l i d o c a i n e and i t s since  -  of  and u r i n e samples r e s p e c t i v e l y ,  The plasma p r o f i l e 24 hours a f t e r  the racemate i s  an o r a l dose i s and R(-) the extent  shown i n F i g u r e 21.  shown i n F i g u r e 22.  enantiomers  i n these  of s t e r e o s p e c f i c  employing t h i s  a d m i n i s t r a t i o n of The u r i n e  profile  The r e l a t i v e  two chromatograms  peak are  disposition.  E f f e c t o f Sodium H y d r o x i d e Treatment on U r i n e C o n t a i n i n g Tocainide-Carbamoyl-O-3-D-Glucuronide It  has been r e p o r t e d (Hasegawa  glucuronides  are u n s t a b l e  readily hydrolysed, tocainide  a l s o forms  the u r i n e , and s i n c e  in alkaline  thus g e n e r a t i n g  solutions  that  and that  the parent compound.  ester they  are  Since excreted  into  the u r i n e assay procedure i n v o l v e s a d d i t i o n of the free  drug,  that no t o c a i n i d e was generated  the assay p r o c e d u r e .  1982)  an e s t e r - t y p e g l u c u r o n i d e , which i s  sodium hydroxide to l i b e r a t e establish  et a l . ,  In order  i t was important  from i t s  to determine  to  glucuronide during  the e f f e c t  of  sodium  FIGURE - 20  CI MASS SPECTRA OF HEPTAFLUOROBUTYRYL DERIVATIVE OF INTERNAL STANDARD, W.49167  375 i  (Mtl)  ee-i 9B-  CH,  83NHCOCHJNHCOCFJCFJCFJ  CH  3  M.W. 374 40-  4 03 (n»!9)  158  Reagent Gas  2BB  :  esB  Methane  i  386  1  i 35S  4 86  458  5BB  55B  -  113 -  T a b l e 11 C a l i b r a t i o n curve d a t a and p r e c i s i o n of assay of plasma u s i n g 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 t a n d a r d , W-49167  q u a n t i t y of each enantiomer (ng)  area r a t i o s ± S.D. S(+)T0C  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  ±0.1454  5.3  2.8292  ± 0.1494  5.2  Correlation coefficient;  RSD(%)  R(- •)T0C  r =  0.9998  0.9998  slope  =  2.7089  2.8240  intercept  = -0.0036  0.0141  The  q u a n t i t y o f i n t e r n a l s t a n d a r d was 1 u g .  RSD(%)  -  114 -  T a b l e 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 u r i n e u s i n g 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 t a n d a r d , W-49167  q u a n t i t y of each enantiomer (ng)  R(+)T0C  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  3.6  area r a t i o s + S . D .  Correlation coefficient,  RSD(%)  r =  0.9999  0.9999  slope  =  1.8074  1.8436  intercept  =  0.0346  0.0372  The  q u a n t i t y o f i n t e r n a l s t a n d a r d was 1 u g .  R(- •)T0C  ± 0.1348  RSD(%)  -  115 -  FIGURE 21  TOCAINIDE ENANTIOMERS plasma profile 24 hours after oral dose of racemate  1. S(+) Tocainide 2.  R(-) Tocainide  3. Internal standard  Li  Blank plasma  _1 10  20 MINUTES  Chromatographic c o n d i t i o n s : 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 ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; make-up gas ( N 2 ) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; c h a r t s p e e t , 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  — '  0  -'  i  10  Blank urine  ——-—i—  20  M IN U T E S  Chromatographic c o n d i t i o n s : 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 ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; make-up gas (N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; c h a r t speed, 0.3 cm/min.  -  hydroxide on p r o d u c t i o n of capillary  -  tocainide  column (15 m x 0.25  detection.  117  i n the u r i n e ,  mm) was used w i t h flame  Diphenhydramine was chosen as  W-49167 was not r e s o l v e d samples c o u l d be analysed base,  eluted  while  the i n t e r n a l  from t o c a i n i d e on an SE-30  from the s h o r t  temperature of  r a t i o s measured are  on t h i s  column.  at 2.6  to 13.0  Area  rather  the i n f l u e n c e  be p a r t i a l l y  the  time of 4  free  minutes  u s i n g a column any t a i l i n g .  The area  Ratio  7.0  0.053  9.5  0.077  10.0  0.084  13.0  0.075  tocainide  different,  i t was assumed  (the  than h y d r o l y s i s , of an a l k a l i ) .  ester glucuronide  i o n i z e d and not e f f i c i e n t l y  tocainide  extracted  into  9.5  sodium  undergoes  to produce a h y d a n t o i n A t pH 7 . 0 ,  from  that no  was produced by the a c t i o n of  h y d r o x i d e on the e s t e r g l u c u r o n i d e  under  as  the area r a t i o s measured between pH v a l u e s r a n g i n g  amount of  cyclization,  Underivatised  Tocainide,  minutes,  since  shown below:  were not s i g n i f i c a n t l y  additional  ionization  phase.  Both peaks d i d not e x h i b i t  pH  Since  fused-silica  the i n t e r n a l s t a n d a r d ,  column w i t h a r e t e n t i o n  standard e l u t e d  160°C.  an SE-30  derivative  (pka 7.8) the  organic  would  118  -  layer.  T h e r e f o r e i t was assumed  alkalinise  urine prior  to l i b e r a t i o n of  -  that  the use  of sodium hydroxide to  to e x t r a c t i o n w i t h dichlororaethane would not l e a d  t o c a i n i d e from i t s  metabolites.  C.  P h a r m a c o k i n e t i c s o f T o c a i n i d e Enantiomers  1.  P h a r m a c o k i n e t i c s o f O r a l T o c a i n i d e Enantiomers The  an  plasma c o n c e n t r a t i o n - t i m e data of each enantiomer, f o l l o w i n g  o r a l dose of 200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e t a b l e t s  h e a l t h y male v o l u n t e e r s are [0.850 for  ± 0.227  R(-)  yg/mL (n=6)  isomer]  volunteers. of  I n Man  f o r S(+)  Peak plasma  isomer and 0.774  (1.26  the o r a l dose.  constant  ug/mL of S(+)  1.52.  In the l a t t e r  in a l l  Plasma l e v e l s  the seven v o l u n t e e r s  TOC (n=7)  By 48 hours  and 0.152  the r a t i o  1.88  ± 0.56  t o c a i n i d e enantiomers constant, 0.75  (h-1)  ± 0.058  the  a t 24 hours  (n=5)  for  the S(+)  plasma  TOC (n=7)] and  [(+)T0C/(-)TOC]  of  the two isomers  i n most v o l u n t e e r s .  When i t  (h-1)  was were was  to 2.53  The pharmacokinetic parameters  to 5.616  the peak  [0.231 ± 0.059  ug/mL of R(-)  are g i v e n i n T a b l e 14.  K a v a r i e d from 3.708  ug/mL  f o r up to 10  of (+)TOC to (-)TOC v a r i e d from 1.53 (n=3).  (n=6)  of each isomer were more or  the plasma c o n c e n t r a t i o n s  v e r y low and could not be measured  a mean of  ug/mL  volunteer (SR),  to others)  the average r a t i o of the two isomers  measurable,  ± 0.234  was reached w i t h i n 45 minutes and i n a n o t h e r ,  ug/mL) was at 3 h o u r s .  hours a f t e r  therefore  levels  were found between 1 to 2 hours i n most of  c o n c e n t r a t i o n remained at h i g h l e v e l (compared  less  seven  However, i n one v o l u n t e e r , a h i g h plasma l e v e l (1.4  each isomer)  level  shown i n T a b l e 13.  to  The a b s o r p t i o n  of o r a l rate  with a mean of 4.29  isomer and from 3.315  to 4.96  with  (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 Time (hra) 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.0 3.0 5.0 7.0 10.0 24.0 30.0 32.0 36.0 48.0 .72.0 *27 hrs •33 hrs  JA  (+)T0C  (-)TOC  0.082 0.356 0.467 0.479 0.498 0.656 0.758 0.697 0.762 0.653 0.563 0.490 0.333  0.086 0.351 0.438 0.439 0.468 0.622 0.681 0.642 0.689 0.584 0.459 0.438 0.262  _  _ _ 0.133 0.083  _  0.087  -  (+)T0C _  SR (-)TOC _  0.355 0.472 0.494 0.535 0.610 0.596 0.563 0.482 0.442 0.407  0.297 0.385 0.426 0.430 0.452 0.485 0.435 0.418 0.369 0.315  0.159  0.085  0.063,  -  -  0.019  _  RE  CK  GK  HF  (+)T0C  (-)TOC  (+)T0C  (-)TOC  (+)T0C  (-)TOC  (+)T0C  (-)TOC  0.129 0.494 0.960 0.825  0.143 0.494 0.933 0.776 0.969  0.146 0.913 0.764 0.845  0.160 0.813 0.712 0.768 -  0.476 1.180 1.402 0.998  0.458 1.116 1.353 0.929  0.888 1.152 1.074 0.699 0.645 0.181 0.106 0.032  0.888 0.758 0.632  0.842 0.802 0.793 0.786 0.768 0.781 0.844 0.629 0.618 0.481 0.234  0.774 0.728 0.708 0.590 0.670 0.696 0.742 0.538 0.508 0.375  0.919 1.105  1.262 1.211 0.512 0.738 0.250  _  0.168  -  0.051 —  -  --  0.556  0.235  0.078  0.152  0.023  --  0.766 0.652 0.526 0.426 0.146  -  0.100 -  -  1.017  -  1.150  1.078  0.519 0.369 0.344 0.301 0.163 0.173  0.912 0.431 0.315 0.300 0.240 0.109 0.105  -  0.160 0.093  -  -  0.111  -  —  -0.071  -  0.028  0.125  (+)T0C  -0.229 0.468 0.600  0.588  (-)TOC  -  0.194 0.427 0.610  -  0.813  0.858  0.493  0.562 0.406  -  0.308 0.231 0.182* 0.119+  -  0.069  -  -  0.263 0.161 0.106* 0.074+  --  Table 14 Pharmacokinetic parameters of t o c a i n i d e enantiomers f o l l o w i n g an o r a l dose of 200 mg (±) tocainide h y d r o c h l o r i d e .  KM K  a  (h  B (h  t  _ 1  _ 1  )  )  l/2(B)  (  h  )  AUC (ug.h mL ) -1  JA  SR  -  (+)T0C (-)TOC  3.708 3.986  4.0934  (+)T0C (-)TOC  0.034 0.042  0.046 0.076  0.068 0.079  RE  CK  HF  4.150 3.315  5.616 4.963  3.907 3.842  0.047 0.067  0.030 0.048  0.041 0.055  GK  0.046 0.063  Mean ± SD 4.29 4.08  ± 0.75 ± 0.82  0.045 0.061  ± 0.012 ± 0.013  (+)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  (+)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  -  mean of 4.08  ± 0.82  (h~ ) (n=4)  121  -  f o r the R(-)  isomer.  This  to an a b s o r p t i o n h a l f - l i f e of 9.6 minutes and 10 minutes and  R(-)  isomers,  respectively.  were 0.045 + 0.012 S(+)  and R(-)  11.9  ± 2.7  their  (h)  isomers.  respectively  B(h) were 16.3  f o r the S(+)  and R(-)  ± 4.0  isomers,  are  3  for (h)  (h-1) the  and  indicating  shown i n F i g u r e  of both the isomers were reached at  different,  S(+)  The l o g plasma c o n c e n t r a t i o n - t i m e  o r a l dose and the a b s o r p t i o n r a t e c o n s t a n t s ,  significantly  2.  (h-1)  f o l l o w i n g an o r a l dose of the racemate  Since peak l e v e l s  not  ± 0.013  The h a l f - l i v e s ,  respectively  f o r the  The d i s p o s i t i o n r a t e c o n s t a n t s ,  and 0.061  differential disposition.  curves  after  (h-1)  corresponded  i t was assumed  that  23.  the same time  K a , were not  the a b s o r p t i o n process was  stereospecific.  P h a r m a c o k i n e t i c s o f I n t r a v e n o u s T o c a i n i d e Enantiomers The  plasma c o n c e n t r a t i o n - t i m e data  f o l l o w i n g an i n t r a v e n o u s  i n f u s i o n of 200 m g ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y male volunteers S(+)  are g i v e n i n Table 15.  and R(-)  isomers  (immediately a f t e r  0.748 ± 0.277 ug/mL and 0.714 enantiomer r a t i o was 1.05 were 0.180 isomers  enantiomer r a t i o infusion.  c o n c e n t r a t i o n s of  the i n f u s i o n was stopped)  ± 0.05.  At 24 h o u r s ,  the mean plasma  ± 0.023 ug/mL f o r the S(+)  and the enantiomer r a t i o was 1.75  increased  to 2.37  Enantiomer r a t i o s  the were  ± 0.300 ug/mL r e s p e c t i v e l y and the mean  ± 0.033 pg/mL and 0.105  respectively  The i n i t i a l  ± 0.39  at 48 hours  at o t h e r times  are  ± 0.35.  levels  and R(-) The  post-intravenous  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 Time 0.33 0.41 0.50 0.75 1.0 1.5 2.0 3.0 5.0 7.0 10.0 24.0 30.0 36.0 48.0 72.0 *32.5 hrs  JA :  SR  RE  CK  (+)T0C  (-)TOC  (+)T0C  (-)TOC  (+)T0C  (-)TOC  (+)T0C  (-)TOC  (+)TOC  (-)TOC  0.635 0.575  0.606 0.538  0.623 0.533  0.551 0.509  0.540  0.495  0.708  0.683  1.233  1.237  0.525  0.467  0.491 0.413 0.447 0.432 0.375  0.425 0.340 0.387 0.327 0.305  0.213 0.179 0.141 0.094 0.045  0.134 0.109 0.072 0.043 0.018  0.540 0.528 0.480 0.515 0.409 0.438 0.319 0.287 0.148 0.129 0.095 0.061  0.527 0.463 0.430 0.463 0.381 0.389 0.282 0.233 0.103 0.072 0.059 0.025  0.542 0.526 0.515 0.531 0.454 0.463 0.368 0.295 0.274 0.162 0.092  0.498 0.489 0.471 0.487 0.407 0.418 0.322 0.228 0.217 0.107 0.057 "  0.636 0.434 0.382 0.372 0.378 0.353 0.287 0.268 0.159 0.121  0.593 0.399 0.347 0.332 0.322 0.278 0.209 0.183 0.069 0.042  1.272 0.709 0.441 0.425 0.421 0.389 0.374 0.367 0.364 0.112 0.074  0.041 0.013  0.041  0.043  1.239 0.694 0.446 0.416 0.416 0.400 0.393 0.389 0.362 0.219 0.147 0.123* 0.083  -  -  -  -  -  -  -  -  -  -  -  -  -  -  —  -  -  -  -  -  -  —  -  -  0.040  Table 16 Enantiomer r a t i o , (+)TOC/(-)TOC, i n the plasma f o l l o w i n g i n t r a v e n o u s i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e to f i v e h e a l t h y male v o l u n t e e r s  KM  JA  SR  RE  CK  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  Time (h)  Mean ± SD  -  The plasma  half-lives  c o n c e n t r a t i o n - t i m e curves  isomers (Table  were 17.05 17).  half-lives  ± 2.5  from the l i n e a r p o r t i o n of the ( F i g u r e 24)  of 5.3  of the two e n a n t i o m e r s .  for  ± 2.4  the S(+)  hours,  v a r i e d between 0.27  to 1.91  isomer and between 0.24  The d i s t r i b u t i o n h a l f - l i v e s , hours (0.81  to 2.16  ± 0.70  hours (0.86  hours)  ± 0.82  the data  Ug/mL) due to the d i f f e r e n c e s characteristics  i n the i n i t i a l  between v o l u n t e e r s .  the  hours)  for  tocainide  the  R(-)  was due to  the  The i n t e r c e p t from 0.132  to  of 1.720  The t e r m i n a l p o r t i o n of the l o g  for a l l subjects  and 0.448 ± 0.033 ug/mL f o r  (AUC) of  to g i v e a ' B '  (0.456  ± 0.034  intercept ug/mL f o r  R(-)tocainide).  P h a r m a c o k i n e t i c parameters c a l c u l a t e d plasma c o n c e n t r a t i o n - t i m e curves  S(+)  distribution  plasma c o n c e n t r a t i o n - t i m e curve was e x t r a p o l a t e d which was r e l a t i v e l y constant  t^/2^a^  for  The l a r g e 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  the os-phase (A) a l s o showed wide v a r i a t i o n ( r a n g i n g  Table  between  concentration-time  c o m p a r a t i v e l y slower d i s t r i b u t i o n i n two v o l u n t e e r s .  S(+)  and R(-)  respectively  hours was observed  The plasma  log  by a computer program (AUTOAN AND NONLIN) and was found to  a two compartment model.  isomer.  -  hours and 11.74  A mean d i f f e r e n c e  was analysed fit  calculated  125  from the area under the  the enantiomers  are  shown i n  18. AUC°° = AUC t + C , . o o t/ 6  Where AUC i s o  the area under the plasma c o n c e n t r a t i o n - t i m e curve from r  time 0 to time t ( t last  = time of l a s t  c o n c e n t r a t i o n measured  c o n c e n t r a t i o n measured).  and 3 i s  C t is  the s l o p e of the t e r m i n a l  the  FIGURE 24  TOCAINIDE ENANTIOMERS PLASMA CONCENTRATION-TIME CURVES AFTER INTRAVENOUS INFUSION OF ( ± ) TOCAINIDE  10  20  30  40  TIME(HRS)  50  60  70  Table 17 Pharmacokinetic parameters of t o c a i n i d e enantiomers f o l l o w i n g an Intravenous i n f u s i o n of 200 mg (±) tocalnde hydrochloride to f i v e healthy male s u b j e c t s  KM  JA  SR  RE  CK  Mean ± SD  (+)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  (+)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  adT ) 1  e l  K  (  h  _  1  )  i/2(e)  t  t  k  l/2(a)  nr  ( h _ 1  >  ( h )  ( 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 11.74  ± 2.5 ± 2.4  (+)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  (+)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 t o c a i n i d e enantiomers f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e to h e a l t h y male s u b j e c t s  AUC (ug.h.mL  l  CLj, (mL/min)  C1 D (mL/min)  (V ) d  Vc  (  (D  g  (L)  Vss  (Vd)  ( L )  (L/kg)  <Vss  ( L / k  ^  )  KM  JA  SR  RE  CK  Mean ± SD  (+)T0C (-)TOC  14.6 9.4  10.9 7.7  9.7 6.8  9.8 5.5  13.6 9.9  11.7 7.8  (+)T0C  114  152  172  169  115  144.4  ± 28.3  (-)TOC  180  217  245  303  167  222.4  ± 54.5  (+)T0C  44  98  68  52  46  61.6  ± 22.4  (-)TOC  53  113  72  58  54  70.0  ± 25.2  (+)TOC  204  226  209  228  199  213  ± 13  (-)TOC  235  213  215  242  185  218  ± 22  (+)T0C  152  164  172  130  45  132  ± 51  (-)TOC  152  178  188  113  47  135  ± 57  (+)T0C  180  179  174  185  136  171  ± 19  (-)TOC  185  193  193  200  149  184  ± 20  (+)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  (+)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  ± 2.2 ± 1.8  -  e l i m i n a t i o n phase. pg.h.mL-1 after  isomer was 7.8  an o r a l dose was much h i g h e r [S(+) R(-)  t o c a i n i d e = 11.72  b i o a v a i l a b i l i t y calculated the S(+)  19).  -  The mean AUC f o r the S(+)  and f o r the R(-)  ug.h.mL-1;  for  129  ±3.56  isomer was 11.7 ± 2 . 2  ± 1 . 8 ug.h.mL-1. t o c a i n i d e = 16.51  pg.h.mL-1].  ± 67.7  percent  The b i o a v a i l a b i l i t y of these isomers  than 100 percent. [S(+)  ± 43.0  percent  f o r the R(-)  isomer  (table  calculated  from the  CO  [X^ ( o r a l ) / X u  t o c a i n i d e = 109.7  the  151.7  CO  c u m u l a t i v e u r i n a r y e x c r e t i o n data  ± 3.76  As a r e s u l t ,  from the plasma data was  isomer and 167.5  The mean AUC  (i.v.),  ± 18.1%;  R(-)  was a l s o h i g h e r tocainide =  112.6].  Table 19 Bioavailability  (%)  KM  JA  of T o c a i n i d e Enantiomers from Plasma Data  SR  RE  CK  Mean ± S . D .  (+)T0C  152.0  109.8  211.1  174.3  111.4  151.7  ± 43.0  (-)TOC)  169.7  92.6  248.7  218.7  108.0  167.5  ± 67.7  Such h i g h o r a l b i o a v a i l a b i l i t y was observed male v o l u n t e e r s dismissed  as  (100-162%) by another group of i n v e s t i g a t o r s  a m e t h o d o l o g i c a l problem ( L a l k a et  no o t h e r r e p o r t e d v a l u e s the present  values  al.,  1976).  but was There  are  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 w i t h which  c o u l d be compared.  In order to ensure observed  i n t h r e e out of four h e a l t h y  that  i n three out of f i v e  the u n u s u a l l y h i g h o r a l b i o a v a i l a b i l i t y s u b j e c t s was not due to an i n c o n s i s t e n c y  -  in  130  the dosage forms used (such as d e g r a d a t i o n  intravenous  solution)  e l e c t r o n capture d e t e c t i o n  that  that  the i n j e c t a b l e  the p l a s t i c  contact  oral  was  the i n f u s i o n .  (Table  it  any drug d u r i n g the T h e r e f o r e , the  i n the amount of t o c a i n i d e  study nor was i t infusion  r o u t e (AUCpg > AUC^-y) was not due  plastic  also 21).  and  after  was assumed short  inconsistency  i n the area under the plasma c o n c e n t r a t i o n - t i m e curve  discrepancies  after  to  i n the dosage form used  for  the  due to l o s s of the drug d u r i n g passage through the  set.  Another p o s s i b l e  reason  t o c a i n i d e may be e n t e r o h e p a t i c indomethacin and s u l i n d a c  for  the h i g h o r a l b i o a v a i l a b i l i t y of  r e c i r c u l a t i o n as r e p o r t e d  (Kwan et  While indomethacin (MW = 357)  molecules  al.,  1976;  f o r drugs  D o b r i n s k a et  and s u l i n d a c  (MW = 356)  of b i l i a r y e x c r e t i o n .  metabolized  (25-40% of dose)  and one would not expect  However, t o c a i n i d e  is  are  enzyme ( b a c t e r i a l  a  known to be  p o l a r i t y and m o l e c u l a r s i z e c o u l d undergo b i l i a r y e x c r e t i o n .  g-glucuronidase  large  the same  to a g l u c u r o n i d e , w h i c h , because of  g l u c u r o n i d e r e a c h i n g the g a s t r o i n t e s t i n a l  such  al.,  and b i l i a r y e x c r e t i o n has been documented, t o c a i n i d e i s  r e l a t i v e l y s m a l l m o l e c u l e (MW = 192) level  the drug  i n the area r a t i o s before  t u b i n g does not adsorb  and i n t r a v e n o u s  1983).  i n f u s i o n set)  s o l u t i o n through the i n f u s i o n s e t ,  time employed f o r  observed  l o s s of  the intended dose was a d m i n i s t e r e d  Since there was no d i f f e r e n c e passing  The p o s s i b l e  (Venocut®  gas  c a p i l l a r y column and  i n j e c t i o n through a d s o r p t i o n by the  the i n f u s i o n set  checked to ensure  fused-silica  (Table 2 0 ) .  c o n t a i n e d i n the i n t r a v e n o u s t u b i n g used f o r  of the drug i n the  both the i n j e c t i o n and t a b l e t s were assayed by  chromatography u s i n g the C h i r a s i l - V a l ®  as  -  its  The  t r a c t may be h y d r o l y s e d by the  enzymes, E - c o l i )  and the  liberated  -  131 -  Table 20 Assay of standard t o c a i n i d e ( 1 . 0 yg) and of samples prepared from t a b l e t s and i n j e c t i o n s  Area R a t i o s Standard  X ± SD  % RSD  Tablets  Injection  (+)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  1.0701  1.1376  1.0489  1.1147  1.0628  1.1537  ±0.0845  ±.0973  ±.0635  ±.0470  ±.0633  ±.0698  8.5  6.0  4.2  5.9  6.0  7.8  -  132  -  T a b l e 21 Assay r e s u l t s  before  and a f t e r the i n j e c t i o n i s the i n f u s i o n set  Area  Ratios  Before  parent  1.9045  1.9453  1.9523  % RSD  1.6  tocainide,  since i t  take p l a c e  f o r drugs  .0314  1.9177 ±  .0314  1.6  i n t o the systemic  f o r an u n s t a b l e  circulation.  e s t e r g l u c u r o n i d e such as  such as  that  is  of readily  c l o f i b r i c a c i d and d i f l u n i s a l , which a l s o  The process of b i l i a r y r e c y c l i n g would be  to predominate a f t e r  o r a l a d m i n i s t r a t i o n when g r e a t e r  the dose passes through the l i v e r , a l t h o u g h s i g n i f i c a n t  recycling  This  i s known t h a t d e c o n j u g a t i o n and r e a b s o r p t i o n  form e s t e r g l u c u r o n i d e s .  2.1  1.8834  1.9166 ±  likely  of  1.8930  X±SD  especially  expected  After  1.9245  drug may be reabsorbed  passed through  c o u l d a l s o occur a f t e r  intravenous  than 90%  biliary  administration.  Plasma C l e a r a n c e Plasma c l e a r a n c e ,  c a l c u l a t e d by d i v i d i n g the i n t r a v e n o u s dose by  the area under the plasma c o n c e n t r a t i o n - t i m e curve f o r each enantiomer, was 144 ± 28 mL/min (S(+)  tocainide)  and 222 ± 54 mL/min  (R(-)  -  tocainide). as  ( T o t a l plasma c l e a r a n c e  166 ± 14 mL/min ( L a l k a et  al.,  1980).  clearance  2.2  133  Patients  al.,  of the racemate has been  1976)  reported  and 194 ± 33 mL/min ( G r a f f n e r  et  w i t h acute m y o c a r d i a l i n f a r c t i o n had a plasma  of 205 ± 64 mL/min ( G r a f f n e r  Renal  -  et  al.,  1980).  Clearance  Renal c l e a r a n c e was c a l c u l a t e d  Cl  =  by the f o l l o w i n g e q u a t i o n :  X (i.v.) ^ AUC ( i . v . ) o  OO  where X^ i s  the cumulative amount of  the parent  drug i n the u r i n e f o r  a  OO  p e r i o d of 96 hours f o l l o w i n g the i n t r a v e n o u s is  Mean r e n a l c l e a r a n c e  the R(-)  isomer,  drug was r e p o r t e d  2.3  as  for  the S(+)  70 ± 25 mL/min.  isomer was 62 ± 22 mL/min and  (Renal c l e a r a n c e  64 ± 6 mL/min by L a l k a et  al.,  of  the racemic  1976).  Volume o f D i s t r i b u t i o n Volume o f d i s t r i b u t i o n , V^ (area) V  and  (i.v.)  the a r e a under the plasma c o n c e n t r a t i o n - t i m e curve f o l l o w i n g the same  dose. for  i n f u s i o n , and A U C q  d(3)  w  a  s  calculated  as  = Dose ( i . v . ) 3 AUC ( i . v . )  the steady s t a t e volume of d i s t r i b u t i o n , e x t r a p o l a t e d  dose s t u d y ,  was c a l c u l a t e d  by:  follows:  from a  single  -  J^t  dose g i v e n by i n t r a v e n o u s  t  d u r a t i o n of  AUMC  area under the f i r s t  where  the t o t a l time vs  time,  f  tocainide  1976; of  2.9  moment curve which i s  t.c.  O  0.49  tocainide  infusion  f o r R(-)  L / k g , Graffner  d i s t r i b u t i o n were 1.98 isomers  S  liters/kg  for  et a l . ,  ± 0.42  T o c a i n i d e Enantiomer L e v e l s i n t h e U r i n e  g i v e n i n Table 22.  (R(-)  L / k g , L a l k a et  ± 0.49  c u m u l a t i v e amount of each isomer e x c r e t e d  isomer  After  was 42.7 of  L/kg for  al.,  volumes the  S(+)  ± 14.2  dose of the racemate (200 mg)  and the R(-)  10.3% between i s o m e r s .  a difference  ± 12.9%  isomer was 32.4  (S(+)  isomer)  10.5% between i s o m e r s .  c o n t r i b u t i o n to e l i m i n a t i o n of  t o c a i n i d e was r e p o r t e d  et a l . ,  1976;  1980.  isomers  excreted  higher  than that  et a l . ,  ± 12.5,  The c o r r e s p o n d i n g v a l u e s  of  Graffner  unchanged i n the  and 31.9  a  after  after  The r e n a l to be 40% ( L a l k a  The cumulative amount of  an i n t r a v e n o u s  an  ± 13.0%  tocainid  unchanged i n the u r i n e f o l l o w i n g an o r a l dose was excreted  i  an o r a l dose, the mean percent e x c r e t i o n of  i n f u s i o n were 42.4  isomer),  ±  respectively.  u r i n e f o l l o w i n g an o r a l and i n t r a v e n o u s  intravenous  t o c a i n i d e and 2.52  The steady s t a t e  L / k g and 2.14  3.  difference  ± 0.22  1980).  R(-)  S(+)  S(+)  (Apparent volume of d i s t r i b u t i o n of  and  The  as  dt + t . c  i n h e a l t h y s u b j e c t s were 1.62  ± 0.2  calculated  area under the product of plasma c o n c e n t r a t i o n -  + 0.43  liters/kg  -  infusion  The mean Vd(3) was 2.47  (±)  134  i n f u s i o n i n four  out  Table 22 Cumulative e x c r e t i o n of t o c a i n i d e enantiomers i n the u r i n e (%) f o l l o w i n g a dose of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e .  Oral  I.V.  dose  Infusion  Bioavailability (%)  KM  JA  SR  RE  CK  HF  GK  (+)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  (+)T0C (-)TOC  39.0 29.8  64.2 52.2  39.5 29.5  29.6 15.7  (+)T0C (-)TOC  85.6 73.8  105.4 100.1  132.9 138.6  103.3 121.6  Mean ± SD 42.7 32.4  ± ±  39.7 32.5  42.4 31.9  ± 12.9 ± 13.0  121.6 128.9  109.7 112.6  ± 18.1 ± 29.9  14.2 12.5  -  of  five  healthy volunteers.  00 from  136  -  As a r e s u l t ,  the b i o a v a i l a b i l i t y  00  the u r i n e d a t a , X o r a l / X i . v . , was h i g h e r u u  these v a l u e s from plasma  were c o n s i d e r a b l y l e s s  However,  than the b i o a v a i l a b i l i t y  the cumulative u r i n a r y e x c r e t i o n vs  26 and 27 are r e p r e s e n t a t i v e  calculated  for  the e x c r e t i o n r a t e vs  are  shown i n Table 23. time p l o t are  plasma d a t a ,  time, and  of a l o g u r i n a r y e x c r e t i o n r a t e vs  time and l o g amount remaining to be e x c r e t e d  vs  than 100%.  data.  F i g u r e 25 shows Figures  calculated  vs  time p l o t as w e l l as  The data f o r  shown i n T a b l e 24.  time p l o t s .  the c a l c u l a t i o n s  involved  the amount remaining to be The h a l f - l i v e s  r a t e p l o t and ARE p l o t ,  The d a t a  calculated  f o l l o w i n g both o r a l and  excreted from  intravenous  a d m i n i s t r a t i o n of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to the same s u b j e c t , shown i n T a b l e 25. tocainide  isomers  Since  are  the plasma c o n c e n t r a t i o n - t i m e data of  have been shown to f i t a two compartment model, no CO  other pharmacokinetic parameters,  other  from the u r i n a r y e x c r e t i o n d a t a .  Enantiomer r a t i o s  following general,  an o r a l dose of  tocainide, 1.57  the racemate are  enantiomer c o m p o s i t i o n of  plasma c o m p o s i t i o n .  At 24 h ,  (n = 7 ) .  ± 0.17  At 48 hours (n = 11)  " ^  rate constant,  was determined as  a  r  calculated  e  i n the u r i n e  parallel  (n = 7)  (+)  In  to that of  tocainide/(-)  w h i l e that of plasma  the r a t i o s were 2.03  ± 0.46  u  shown i n table 26.  the u r i n e runs  the u r i n e and 1.81 ke,  a n a  the enantiomer r a t i o ,  i n the u r i n e was 1.41  ± 0.24  than t ^ ^  i n the plasma.  ± 0.53  (n = 7)  is  the f r a c t i o n of  follows:  the drug e x c r e t e d  in  The u r i n a r y e x c r e t i o n  k e = KE x f  where ' f  was  unchanged i n the u r i n e  -  £37'*-  FIGURE 25  CUMULATIVE AMOUNT OF SWTOCAINIDE E X C R E T E D IN THE URINE  20  40  60 TIME(h)  SUBJECT:  CK  80  -  138  -  FIGURE.26  SEMILOG ARITHMIC PLOT OF EXCRETION RATE VERSUS TIME AFTER INTRAVENOUS ADMINISTRATION OF (OTOCAINIDE HYDROCHLORIDE  4000,  r 1000 S(+) tocainide - 0.9428 •1 6 » 0.0391 hr' tlj = 17.7 hrs  "tt  T  I  O—o  R ( - ) tocainide T c 0.9518 1 g » 0.0463 hr tH • 14.9 hrs  U X 100  101  20  40  80  60 TIME(h)  SUBJECT:  CK  -  139 -  FIGURE 27  AMOUNT  REMAINING TO BE E X C R E T E D OF  TOCAINIDE  AFTER  INTRAVENOUS  ADMINISTRATION  HYDROCHLORIDE  40  o \  «  %  •—•  O—O  10  S(+) tocainide r » 0.9948 B « 0.0481 h" t>s - 14.4 hrs  1  R ( - ) tocainide r = 0.99S4 8 " 0.0601 h" th - 11.5 hrs  1  cn  £ X  3  i 8 3 X  10  •11  . 20  ,  ,  _  40  60  80  TIME(h)  SUBJECT:  CK  Table 23  Dose: l . v . I n f u s i o n  collection time (h) 1.0 2.0 3.0 5.0 7.0 9.0 11.0 12.5 13.5 19.0 21.0 24.0 27.0 28.0 30.0 32.5 37.0 43.0 46.0 53.0 58.0 63.0 68.0 72.0 78.0 82.0 86.0 96.0  plotting time (h)  0.5 1.5 2.5 4.0 6.0 8.0 10.0 11.7 13.0 16.2 20.0 22.5 25.5 27.5 29.0 31.2 34.7 40.0 44.5 49.5 55.5 61.5 65.5 70.0 75.0 80.0 84.0 91.0  U r i n a r y e x c r e t i o n data for the rate p l o t . of 200 mg (±) t o c a i n i d e h y d r o c h l o r i d e at a r a t e of  total (+)T0C  total (-)TOC  volume (mL)  PH  (pg)  (Ug)  98.0 46.0 73.0 540.0 473.0 296.0 210.0 386.0 102.0 445.0 74.0 60.0 446.0 514.0 126.0 56.0 130.0 296.0 122.0 338.0 406.0 192.0 360.0 150.0 164.0 160.0 240.0 410.0  6.8 5.6 5.5 6.5 6.0 6.3 6.8 7.3 6.6 5.8 5.3 5.9 5.5 6.3 5.5 5.2 5.3 5.6 5.4 6.8 7.0 6.2 6.0 6.0 5.2 5.2 5.3 5.2  2886 1989 2638 3068 3802 2371 1374 824 421 4860 1350 763 1772 635 446 933 1399 2995 1432 774 377 588 669 414 122 303 178 325  3010 2074 2548 2913 3424 2242 1108 668 337 4249 996 599 1170 241 277 668 870 2450 853 386 123 323 294 235 49 120 72 179  10 mg/mln.  excretion r a t e (+)T0C (Pg/h) 2886 1989 2638 1534 1901 1186 687 549 421 884 675 254 591 635 223 373 311 499 477 111 76 118 134 103 31 75 44 32  excretion r a t e (-)TOC (Pg/h) 3010 2074 2548 1456 1712 1121 554 445 337 772 498 199 390 241 138 267 193 408 284 55 25 65 59 59 8 30 18 17  i -  S  i  -  141 -  Table 24  Dose:  Time (hrs) 0- 0 0- 1 1- 2 2- 3 3- 5 5- 7 7- 9 9-11 11-12.5 12.5-13.5 13.5-19 19-21 21-24 24-27 27-28 28-30 30-32.5 32.5-37 37-43 43-46 46-53 53-58 58-63 63-68 68-72 72-78 78-82 82-86 86-96  U r i n e data f o r ARE p l o t of S(+) t o c a i n i d e IV i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e at a r a t e of 10 mg/min.  Amount remaining to be e x c r e t e d (mg)  amount e x c r e t e d (mg)  cumulative amount (mg)  -  -  40.70  2.886 4.875 7.513 10.581 14.383 16.754 18.128 20.373 25.233 25.233 26.583 27.346 29.118 29.753 30.199 31.132 32.531 35.526 36.958 37.732 38.109 38.697 39.366 39.780 39.902 40.205 40.383 40.708  37.81 35.82 33.18 30.12 26.31 23.94 22.57 20.32 15.46 15.46 14.11 13.35 11.58 10.94 10.50 9.56 8.16 5.17 3.74 2.96 2.59 2.00 1.33 0.92 0.79 0.49 0.31 0.00  2.886 1.989 2.638 3.068 3.802 3.802 1.374 0.421 4.360 4.860 1.350 0.763 1.772 0.635 0.446 0.933 1.399 2.995 1.432 0.774 0.377 0.588 0.669 0.414 0.122 0.303 0.178 0.325  .  (X~)  Table 25 H a l f - l i f e (hours) of tocainide enantiomers c a l c u l a t e d plasma and urine data  from  KM  JA  SR  RE  CK  HF  GX  16.8 12.6  14.8 12.6  Mean * SD  .oral  (+)TOC (-)TOC  19.9 16.1  14.9 9.1  10.2 8.7  14.5 10.3  22.8 14.4  16.3 ± 4.0 11.9 ± 2.7  •i.v.  (+)TOC (-)TOC  20.8 15.3  17.1 11.4  14.1 10.1  15.5 9.2  17.7 12.7  17.0 ± 2.5 11.7 ± 2.4  i_oral  (+)T0C (-)TOC  14.1 11.4  11.3 8.5  13.4 10.9  18.6 13.5  15.6 13.7  14.6 ± 2.7 11.6 ± 2.1  (+)T0C (-)TOC  19.1 15.6  11.6 10.0  16, 12.  20.6 12.3  17.7 14.9  17.1 ± 3.4 13.0 ± 2.2  i—oral  (+)TOC (-)TOC  10.8 9.6  7.5 6.1  11.5 9.7  13.2 10.6  11.1 12.1  10.8 ± 2.0 9.6 ± 2.2  l-i.v.  (+)TOC (-)TOC  15.9 13.8  10.7  13.7 8.6  16.3 11.9  14.4 11 .5  14.2 ± 2.2 11.4 ± 1.8  Plasma  Urine  Rate P l o t • i.v.  ARE Plot  Table 26 Enantiomer r a t i o , (+)T0C/(-)TOC, in the plasma and urine f o l l o w i n g an o r a l dose of 200 mg ( t ) t o c a i n i d e hydrochloride to 7 healthy male v o l u n t e e r s .  JA  KM i LIUC  SR P  RE  U*  P  U  -  1.01  -  1.19  -  0.91  -  1.00  1.09  1.04  1.16  1.02  1.06  2.0  1.08  1.04  1.29  1.04  3.0  1.10  1.13  1.15  5.0  1.12  1.19  7.0  1.23  10.0  (h)  P*  0.25  0.95  0.50  U  P  CK u  P  GK  HF  U  P  U  P  U  -  -  1.09  -  1.18  -  1.12  -  1.06  -  1.02  1.10  1.05  1.07  1.01  1.10  1.03  0.98  0.92  1.03  1.16  1.16  1.09  1.11  0.98  1.14  1.12  1.04  0.94  0.99  1.09  1.04  1.16  1.18  1.20  1.58  1,17  1.15  1.21  0.95  1.20  1.15  1.13  1.05  1.20  1.26  1.17  1.09  1.22  1.15  1.31  1.29  1.22  1.16  1.18  -  1.21  1.15  1.12  1 .28  1.17  -  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  1.61  2.50  2.36  1.62  1.39  72.0  -  2.97  2.28  -  2.00  1.50  -  3.79  -  1.50  *P " plasma U = urine  -  0.90  0.88  -  2.75 3.09  1.04  -  0.96 0.96  -  (which i s  .  the o v e r a l l e l i m i n a t i o n r a t e  The mean u r i n a r y e x c r e t i o n r a t e c o n s t a n t ,  t o c a i n i d e was 0.0337 ± 0183 h - 1  -  determined from % X ) and K „ i s u ti  constant.  h  144  - 1  and f o r  the R(-)  The n o n - r e n a l e l i m i n a t i o n r a t e c o n s t a n t ,  metabolic e l i m i n a t i o n , i s  ke  f o r the  isomer 0.0386 ± 0.0229  knr,  including  given by: knr  = K E (1 -  f).  There was wide v a r i a t i o n i n the v a l u e s  of k^- i n d i f f e r e n t  (0.0492 ± 0.32  0.0920 + 0.0586 h ~ 1 f o r  isomer) values  h " 1 for  tocainide,  of n o n - r e n a l e l i m i n a t i o n r a t e constant  (table  because of i t s  f a s t e r metabolic  Cl.p, AUC, k ^ volunteers  f o r the R(-)  R(-) The h i g h  isomer  was  (glucuronidation?)  i n pharmacokinetic parameters such as  c l e a r l y points  absorption,  of t o c a i n i d e  may be due to d i f f e r e n c e s  i n the a b s o r p t i o n ,  assumed that  the s t e r e o s e l e c t i v i t y is  it  is  i s most l i k e l y  Differences  Since no  d i s t r i b u t i o n and e x c r e t i o n , i n the proces's-of  known to be m e t a b o l i z e d  the g l u c u r o n i d a t i o n process i s drugs,  in healthy  i n the r a t e of  d i s t r i b u t i o n , metabolism or e x c r e t i o n .  tocainide  t]_/2»  to a s t e r e o s e l e c t i v e d i s p o s i t i o n .  was observed  chiral  i n metabolism.  elimination  between the two enantiomers  stereoselectivity  Since  subjects  17). The d i f f e r e n c e s  in  S(+)  presumably because of the d i f f e r e n c e s  expected  S(+)  difference it  is  metabolism.  to a g l u c u r o n i d e and  since  known to be s t e r e o s e l e c t i v e f o r many that g l u c u r o n i d a t i o n i s  selective  step i n v o l v e d i n the d i f f e r e n t i a l  isomers.  Figures  the  d i s p o s i t i o n of  28a and 28b show the r e l a t i v e  proportions  stereotocainide of b o t h  -~145  -  FIGURE 2 8 - '  URINE P R O F I L E B E F O R E ( A ) HYDROLYSIS  AND A F T E R ( B )  WITH ^ - G L U C U R O N I D A S E 1.S(+)tocalnide 2.R(-)tocainide 3.Internal s t a n d a r d  Chromatographic c o n d i t i o n s : 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 ; d e t e c t o r (ECD) t e m p e r a t u r e , 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas (N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; c h a r t speed, 0.3 cm/min.  -  isomers  146  -  i n the same u r i n e sample before  3-glucuronidase  enzyme ( S o u r c e :  isomer generated  after  isomer showing that  and a f t e r  The amountof  R(-)  h y d r o l y s i s was h i g h e r than that of the  S(+)  the R(-)  bov ine  liver)•  h y d r o l y s i s with  t o c a i n i d e g l u c u r o n i d e was i n excess i n the  urine.  4.  Chromatographic A n a l y s i s o f Uremic Plasma E x t r a c t on C h i r a s i l - V a l Fused-silica  C a p i l l a r y Column (50 m x 0.31 mm)  F i g u r e 29 i s number of of  29). ure,  the blank uremic plasma p r o f i l e  peaks due to endogenous  the endogenous  analysis  of R(-)  compounds e l u t i n g at 8.7  (He)  flow r a t e ,  considered  impossible  uremic plasma  columns of d i f f e r e n t  the plasma  tocainide  to use t h i s  samples.  column temperat-  c a r r i e r gas,  At t h i s  isomer and t h e r e f o r e  c a p i l l a r y column f o r  p o l a r i t y to r e s o l v e  from t o c a i n i d e  isomers.  It  the endogenous  as  were e x t r a c t e d ,  with  tocainide  Six serum samples c o n t a i n i n g c r e a t i n i n e  to 16.8 mg%, o b t a i n e d derivatised  serum  16.8 mg% d i d not show  peaks i n the chromatogram that would i n t e r f e r e  the range of 4.9  was  was known from our own e x p e r i -  h i g h as  (Figure 30).  it  component i n  samples c o n t a i n i n g c r e a t i n i n e l e v e l s  determinations  endo-  time i t was decided to t r y a combination  c a p i l l a r y column that  extra  or  the assay of  ments w i t h a carbowax 20 M f u s e d - s i l i c a  any  (Fig.  d i d not improve the r e s o l u t i o n o f t h i s  genous compound from the R(-)  One  minutes d i d i n t e r f e r e with-'the  the H2 as  use of  large  i n the plasma.  isomer which had an i d e n t i c a l r e t e n t i o n time  temperature-programming  of  compounds present  showing the  Changing the chromatographic c o n d i t i o n s such as c a r r i e r gas  8  in  from Vancouver General H o s p i t a l ,  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 c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (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 ( H 2 ) f l o w , 1 mL/min; make-up gas (N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; c h a r t 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 (50 m x 0.2 mm)  Chromatographic c o n d i t i o n s : 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). I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas ( a r g o n : methane, 9 5 : 5 ) , 60 mL/min; column p r e s s u r e , 1 9 0 ° C ; s p l i t vent f l o w , 30 mL/min; c h a r t speed, 0.3 cm/sec.  - 149 -  column.  None of  a carbowax  these samples e x h i b i t e d  20 M f u s e d - s i l i c a  capilary  in series with a C h i r a s i l - V a l ®  resulting  chromatogram  The  chromatogram  of  shown i n F i g u r e  chromatogram,  was obtained tion.  for  Therefore,  resolution  the plasma c o l l e c t e d all  mm) was  m x 0.31  mm).  further  determination.  The  of  the  from a p a t i e n t  analyses,  the  two  A very  enantiomers,  with renal  dysfunc-  two columns connected  s e r i e s were used ( F i g u r e 3 1 ) .  C a l i b r a t i o n d a t a and p r e c i s i o n of  assay u s i n g  column i s  5.  a  31.  with tocainide  with excellent  for  column (50  Therefore  the blank uremic plasma d i d not c o n t a i n any  e x t r a peaks that would i n t e r f e r e clean  peaks.  column (20 m x 0.2  connected  is  interfering  the d u a l c a p i l l a r y  shown i n T a b l e  P h a r m a c o k i n e t i c s o f T o c a i n i d e Enantiomers  in  plasma  27.  i n Renal D y s f u n c t i o n and  During Hemodialysis I n view of dose as its  the e x t e n t of u r i n a r y e x c r e t i o n  (25-40% of d o s e ) ,  tocainide  Besides  renal  the e f f e c t  of  of r e n a l  certain  the pharmacokinetic  insufficiency  on e l i m i n a t i o n of  to permit r a t i o n a l  physiological  dosing  characteristics  adjust-  changes  For example,  the  decreased due to changes i n  ( G i b a l d i and P e r r i e r ,  M o d i f i c a t i o n of e r y t h r o c y t e  volume of d i s t r i b u t i o n of  and anatomical  parameters of d r u g s .  volume of d i s t r i b u t i o n of d i g o x i n i s distribution  and  the r i s k of accumulation of drugs and t o x i c m e t a b o l i t e s  insufficiency,  also alter  1973).  (40%  c a r b a m o y l - O - 0 - D - g l u c u r o n i d e (TOCG)  these substances should be q u a n t i t a t e d  in  tocainide  i n t a c t drug i n normal s u b j e c t s and 20-70% i n d i s e a s e s t a t e s )  major, m e t a b o l i t e ,  ment.  of  concentrations  some d r u g s .  Anemia i s  1972;  Reuning e t  can a l s o d i s t u r b  accompanied  by a  al., the  -  150  -  FIGURE 31 GC/ECD PROFILE OF UREMIC PLASMA 2 HOURS AFTER INTRAVENOUS INFUSION OF ( ± ) TOCAINIDE TO AN ANEPHRIC PATIENT A. B.  CHIRASIL-VAL® FUSED-SILICA CAPILLARY COLUMN CARBOWAX 20 M AND CHIRASIL-VAL®  B  iOCN  COCO  Chromatographic c o n d i t i o n s : A . I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (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 l o w , 1 mL/min; make-up gas (N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 151.7 k P a ; c h a r t s p e e d , 0.3 cm/min. B. Oven temperature 2 0 0 ° C ; column i n l e t p r e s s u r e , 179.3 k P a . Other c o n d i t i o n s 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 u s i n g 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).  q u a n t i t y of each enantiomer (ng)  S(+)T0C  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  2.1040 ±  6.0  2.1077 ± 0.1131  5.3  1000.0  area r a t i o  .1264  mean RSD ± SD  Correlation coefficient,  RSD(%)  4.2  ±  R(- •)T0C  1.4  4. 7 ± 0.8  r =  0.9988  0.9996  slope  =  2.0889  2.0951  intercept  = -0.0151  -0.0042  The q u a n t i t y of i n t e r n a l s t a n d a r d was 1 u g .  RSD(%)  -  decreased plasma  152  b i n d i n g of g e n t a m i c i n to red blood c e l l s  concentrations  hemoglobin l e v e l s changes caused t i o n of drugs  alterations phenytoin,  (Riff  and J a c k s o n ,  given o r a l l y ( P e t i t p i e r r e ,  serum c o n c e n t r a t i o n s  In  of t o t a l  ites.  is  1974).  1967).  treated  T h i s unusual phenomenon  who  al.,  i n uremia.  the f ormation of the m e t a b o l -  uremia decreases a c e t y l a t i o n of s u l f i s o x a z o l e 1969;  Ogg et  al.,  are shown i n t a b l e 28.  had been undergoing d i a l y s i s concentration,  1968).  infusion  with renal  The s u b j e c t was a 22 year o l d male  three times a week f o r  13.4 mg%).  24 hours f o l l o w i n g drug i n f u s i o n ,  and  Enantiomers  200 mg of ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t  creatinine  its  (Melk et  a t t a i n s r e l a t i v e l y h i g h serum l e v e l s  Pharmacokinetic o f Intravenous Tocainide  dysfunction  with  lower than i n normal  Plasma c o n c e n t r a t i o n - t i m e data f o l l o w i n g an i n t r a v e n o u s of  In  causes  r a t e of p h e n y t o i n to  p - a m i n o s a l i c y l i c a c i d (Reidenburg et a l . ,  5.1  absorp-  E l i m i n a t e d p r i m a r i l y i n the  r e n a l d i s e a s e decreases  F o r example,  drug are  decreased.  the  Fabre et a l . ,  ( 5 - p h e n y l - 5 - p - O H - p h e n y l hydantoin)  t h i s metabolite  other c a s e s ,  to some degree,  In uremic p a t i e n t s  transformation  O d a r - C e d e r l o f and Borga,  urine,  F u n c t i o n a l and a n a t o m i c a l  1972;  to the metabolism of d r u g s .  from an i n c r e a s e d  w i t h normal  renal insufficiency occasionally  s u b j e c t s and the plasma h a l f - l i f e  1970;  1971).  by c h r o n i c uremia may a f f e c t ,  p r i n c i p a l metabolite  and consequently  become much h i g h e r than i n p a t i e n t s  a d d i t i o n to these changes,  results  -  at  two years  (serum  Blood samples were c o l l e c t e d up to the 25th h o u r , h e m o d i a l y s i s  was  s t a r t e d and the d i a l y s i s  and blood sampling c o n t i n u e d f o r 5 h o u r s .  Even  though the plasma l e v e l s  were lower than i n h e a l t h y i n d i v i d u a l s who have  -"153  -  Table 28 Plasma c o n c e n t r a t i o n - t i m e data f o l l o w i n g i n t r a v e n o u s i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t with r e n a l d y s f u n c t i o n .  Time (h)  (+)T0C (yg/mL)  (-)TOC (ug/mL)  ratio (+)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,  failure,  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  levels  on repeated  half-life  the plasma c o n c e n t r a t i o n s p e r s i s t e d  d o s i ng to p a t i e n t s  calculated  tocainide  with kidney d i s e a s e .  from the l i n e a r p o r t i o n of the l o g  c o n c e n t r a t i o n time curve ( F i g u r e 32) was 45.3 hours f o r and 28.7  hours f o r the R(-)  These v a l u e s  isomer  correspond to 2.5  compared to normal s u b j e c t s . half-lives  r a n g i n g from 16.6  of 9 p a t i e n t s mL/min). for  with t o t a l  Oltmans et a l . ,  the racemate  dysfunction. mL/min f o r  contribution  the S(+)  This is  compared to the  consistent is  clearance < 5  a h a l f - l i f e of 8.5  ± 4.6  with renal dysfunction.  is  isomer and 146  the r e n a l  significantly 8 of the  increased S(+)  isomer was 365 l i t e r s . hours f o r  Wieger et a l .  hours for  and r e n a l  to a r e d u c t i o n of 40% from  The ( V j )  the h a l f - l i f e was 6.5 isomer.  the S(+)  hours  approximately 40% i n h e a l t h y  and that of R(-)  the R(-)  i n a group  with the o b s e r v a t i o n t h a t  i n healthy subjects.  5 hour h e m o d i a l y s i s ,  as  (1983) a l s o observed a h a l f - l i f e of 57.4  clearance  isomer was 338 l i t e r s  isomer  in h a l f - l i f e  the racemate  (creatinine  isomer which corresponds  to t o t a l  5.4 hours f o r  plasma  (1983) have r e p o r t e d  hours f o r  The volume of d i s t r i b u t i o n ( V a )  was s i m i l a r  increase  Plasma c l e a r a n c e was 86 mL/min f o r  normal s u b j e c t s .  infusion,  fold  renal failure  toxic  29).  Wiegers et a l . , to 42.7  to  The  i n a patient with active hepatic necrosis  the R(-)  subjects.  (Table  in renal  the S(+)  During a isomer and  (1983) a l s o have observed  the racemate  i n a group of 9  patients  The change i n enantiomer c o m p o s i t i o n with time  to the o b s e r v a t i o n  the enantiomer r a t i o ,  i n healthy subjects. (+)  T 0 C / ( - ) TOC, was  At 24 hours 1.35.  post  - • 155^ -  FIGURE 32  LOQ PLASMA CONCENTRATION-TIME CURVE FOLLOWING INTRAVENOUS INFUSION OF 200 MQ OF (t)TOCAINIDE HYDROCHLORIDE TO A PATIENT WITH RENAL DYSFUNCTION.  S(+)tocainide r=0.9810 ^=0.0153 hr" t^=45.3 h r s  E a  a  R(-)tocainide r=0.9691 0.0241 h r " .75 h r s  UJ o  z  o u  < 2 CO <  10  15  TIME (HRS)  20  25  Table 29 Pharmacokinetic parameters of t o c a i n i d e enantiomers f o l l o w i n g an i n t r a v e n o u s i n f u s i o n of 200 mg ( ± ) t o c a i n i d e h y d r o c h l o r i d e to a p a t i e n t with r e n a l d y s f u n c t i o n and d u r i n g 5 hr h e m o d i a l y s i s .  Patient  AUC (ug.h.raL t  l/2(g)  C1T (Vd)  (  h  r  s  l  )  )  (mL/min) (L)  p  (Vd)g  (L/kg)  t  l/2  ^  n r s  fraction  ^ during  dialysis  removed by  % removed by  (-)TOC)  19.29  11.35  11.7  + 2.2  45.3  28.7  17.05  + 2.5  86.0  146.0  144.0  + 28  222.0  + 54  338.0  365.0  213.0  + 13  218.0  + 22  20.0  dialysis  dialysis  2.47  4.86 ±4.8  49.8  (+)T0C)  (n=5)  (+)T0C)  4.50  C 1 D (mL/min)  Healthy subjects  ± 16.3  + 0.43  (-)TOC) 7 .8 11.7  2.52  + 1 .8 + 2.4  + 0.49  -  -  6.5  5.4  -  -  0.353  0.385  -  -  -  -  35.0  38.0  -  5.2  157  -  D i s p o s i t i o n o f Drug G l u c u r o n i d e s i n R e n a l F a i l u r e Glucuronide conjugation  patients with renal  failure.  has  not been e x t e n s i v e l y  investigated  S t u d i e s i n r a t s have shown t h a t  g l u c u r o n i d a t i o n process may be d i m i n i s h e d (p-aminobenzoic increased  (naphthol)  or u n a f f e c t e d  mental r e n a l f a i l u r e Leber et a l . ,  (Canalese et  1972).  Recent  of  1979;  Gugler,  glucuronides  regeneration  conjugates i n r e n a l f a i l u r e  1979a; Gugler et  by the k i d n e y s  al.,  1979b).  biliary floral  excretion,  the g l u c u r o n i d e s glucuronides  ^-glucuronidase  reabsorptdLon 1971;  true of  of  Hartiala,  carried  of  1973).  particularly  liver,  (Levy and C o u c h i e , 1966; g l u c u r o n i d e may l e a d a c c u m u l a t i o n of  parent  the a c t i v e  of  compared  of which i s  2 to 6 - f o l d  diflunisal,  Following  the m i c r o -  can a l s o be present i n most  and i n t e s t i n a l Failure  longer  (Gugler et  al.,  epithelium  to e x c r e t e a leading  to  An example  an e s t e r - t y p e g l u c u r o n i d e , in renal f a i l u r e al.,  Another example of a drug which forms is  not  permitting  compound i n r e n a l f a i l u r e .  to h e a l t h y s u b j e c t s  glucuronides  tract,  the c o n j u g a t e ,  p r o v i d e d by c l o f i b r i c a c i d which forms half-life  is  i n t o the b i l e .  the g l u c u r o n i d e s  1970).  and  irrever-  compound ( O k o l i c s a n y i et  kidney, spleen  to h y d r o l y s i s  parent  Although e x c r e t i o n  this  (3-glucuronidase which i s  Wakabayashi,  1979;  (Faed  may become s u b s t r a t e s f o r  H y d r o l y s i s of  out by the lysosomal  tissues;  excreted  the g a s t r o i n t e s t i n a l  the l i b e r a t e d  experi-  of  i n t o the u r i n e r e s u l t s i n the  s i b l e e l i m i n a t i o n of the conjugates from the body, necessarily  in  Howie and Bourke,  reports indicate  drug from c e r t a i n g l u c u r o n i d e McQueen,  1980;  the  acid),  (4-methylumbelliferone) al.,  in  is the  patients  1979b). e s t e r - and  a r e c e n t l y developed  ether-type  salicylate  analgesic.  -  In  a recent  clearance renal  s i n g l e dose s t u d y ,  -  was shown that  the t o t a l  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  failure  (De Schepper et a l . ,  P o s s i b l e mechanisms renal  it  158  failure,  include altered  1977;  body  in patients  Verbeeck et a l . ,  1979).  g l u c u r o n i d a t i o n of d i f l u n i s a l  b i l i a r y e x c r e t i o n of one or both of the  f o l l o w e d by h y d r o l y s i s i n the i n t e s t i n a l  tract  and r e a b s o r p t i o n  deconjugation  glucuronides  f o r the e s t e r g l u c u r o n i d e of  r a t e (DeSchepper Since increased  et a l . ,  1977;  L e v y , 1979;  (2.5  fold)  indicate  5.3  are  increased  the  with r e n a l  stereospecificity  The present  the o c c u r r e n c e o f s t e r e o s p e c i f i c i t y  study a l s o does  since  to the same extent  of  the h a l f - l i v e s  (Table  of  29).  Hemodialysis Hemodialysis i s  waste m a t e r i a l flow,  dialysate  the most common method of removing  i n chronic renal f a i l u r e  to decrease the extent characteristics  patients.  Factors  flow and s u r f a c e area of the d i a l y s i s  influence d i a l y s a b i l i t y .  A decrease i n each of  to which a drug i s  of a drug w i l l  such as  that  blood  will  these f a c t o r s w i l l  dialysed.  Those drugs  endogenous  membrane  tend  The pharmacokinetic  a l s o have a s i g n i f i c a n t  a b i l i t y of a drug to be d i a l y s e d . of  i n the p a t i e n t  in renal insufficiency.  both the isomers  clofib-  1980).  There has not been any r e p o r t about  drug d i s p o s i t i o n not  Faed,  the  of the parent drug may be due to one or  more of the above mechanisms o p e r a t i n g dysfunction.  of  of one or both of  t o c a i n i d e a l s o forms an e s t e r - t y p e g l u c u r o n i d e ,  half-life  in  glucuronides  unchanged d i f l u n i s a l , and systemic as has been d e s c r i b e d  with  impact on the  have a l a r g e volume  d i s t r i b u t i o n or are h i g h l y p r o t e i n bound tend to be p o o r l y d i a l y s e d .  -  The use  of b i n d i n g and d i s t r i b u t i o n data w i l l  a p p r e c i a t i o n of estimates  of  g i v e one a g e n e r a l  the d i a l y s a b i l i t y of a g i v e n d r u g , but more  the amount removed may be d e s i r a b l e  adjustments.  One approach i n v o l v e s  during d i a l y s i s , ti/2»  159 -  (tj/2)d,  The f r a c t i o n of  f  the purpose  the use of h a l f - l i v e s  and when d i a l y s i s  -  ^ i n h  (  1  _  e  of  of dose  drugs  i s not being performed,  the drug removed by d i a l y s i s ,  h n  =  for  precise  -°-  6 9 3  t / ( t  f,  is  i/2  )  g i v e n by  d )  '1/2 where t i s  the d u r a t i o n of d i a l y s i s  a drug that w i l l  ( G w i l t et  be removed by d i a l y s i s ,  al.,  1978).  The amount of  X ^ , can be c a l c u l a t e d  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  if  c o n c e n t r a t i o n , C are  the  known.  Xd = f.cv T h i s amount can then be r e p l a c e d a f t e r  dialysis.  T o c a i n i d e i s not p r o t e i n bound to any g r e a t extent ( E l v i n et a l . , about (25  1982;  ± 14%)  of  Sedman et a l . ,  1982)  (about  10%)  and i t had been shown that  the drug was removed by d i a l y s i s  for 4 hours  ®  (parallel-plate a r e a , , 1.2 mL/min)  dialyser,  the present  enantiomers  study i n one p a t i e n t ,  by s u b s t i t u t i n g (S(+)T0C, t i / i  The amount of S(+)  flow of 550  the f r a c t i o n of  that would be removed by d i a l y s i s  equation:  equation:  (surface  1983).  calculated  hrs).  w i t h c e l l u l o s e membrane,  m ) blood flow of 150-250 mL/min and a d i a l y s a t e  (Wiegers e t a l . , In  ER - 8 5 ,  was 0 . 3 5 3 , which was  the f o l l o w i n g h a l f - l i f e v a l u e s =  ^  5  «  3 h r s  »  ^1/2^  tocainide  =  6  ,  5  n  r  s  a  isomer removed was c a l c u l a t e d  n  i n the d  1  =  above  5  by the f o l l o w i n g  -  XD  160 -  = 0.353 x 0.204 x 338 = 24.113 mg  T h i s corresponds  dialysis.  The p r e d i a l y s i s  plasma was 0.204 was 338  to removal of 35% of  the drug i n the body j u s t  c o n c e n t r a t i o n of S(+)  isomer i n the  ug/mL, and the volume of d i s t r i b u t i o n of  this  before  arterial isomer  liters. Similarly,  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 C a l c u l a t i o n s During Hemodialysis The  log a r t e r i a l  plasma c o n c e n t r a t i o n - t i m e curve obtained d u r i n g  the 5 h h e m o d i a l y s i s i s The  dialysis  shown i n F i g u r e 3 3 .  c l e a r a n c e was c a l c u l a t e d  DP  c i ™  where 0 1 ^ = d i a l y s i s  plasma  =  p  xQ  A  by the f o l l o w i n g f o r m u l a :  CA A  clearance  = c o n c e n t r a t i o n of drug i n a r t e r i a l = c o n c e n t r a t i o n of drug i n venous Qp  = plasma flow r a t e  plasma  plasma  through the d i a l y s e r  = Q b (1 - H) where  = blood flow r a t e H  = hematocrit. The  of  f o l l o w i n g t a b l e shows  both the isomers  of  the a r t e r i a l  and venous  t o c a i n i d e and the A-V d i f f e r e n c e  concentrations (Table  30).  -161  FIGURE 33  LOG  ARTERIAL P L A S M A C O N C E N T R A T I O N - T I M E C U R V E DURING 5HR HEMODIALYSIS  •  a.  S(+)tocainide r=0.9626 fi =0.1055 h -1 r t, =6.5 hrs h  A—A R(-)tocainide -1  < cc  KZ UJ  o z o o  < CO  < _l  OL  TIME  (HRS)  Table 30 Arterial  Dialysis time (h)  and venous c o n c e n t r a t i o n s of  the isomers of t o c a i n i d e and the A-V  arterial  difference.  venous  A-V  difference  (+)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  .0174  .0441  0.1150  0.0671  -  At 13.7, Qp  -  a blood flow r a t e of 200 mL/min and a h e m a t o c r i t v a l u e of  the plasma  flow r a t e i s  and A-V d i f f e r e n c e s  clearances  172.6 mL/min.  Substituting  the v a l u e o f  i n the above e q u a t i o n the f o l l o w i n g  dialysis  were o b t a i n e d . Dialysis  Time  (hrs)  C l e a r a n c e (mL/min)  (+)T0C  (-)TOC  1.0  14.5  37.6  2.0  23.0  43.5  5.0  22.7  68.4  Mean ± SD  6.  163  20.0  ± 4.8  49.8  ± 16.3  S t e r e o s e l e c t i v e S a l i v a r y E x c r e t i o n o f T o c a i n i d e Enantiomers i n Man None of the p u b l i s h e d s t u d i e s on t o c a i n i d e has examined  p o s s i b i l i t y of saliva.  s e c r e t i o n of R , S - t o c a i n i d e or i t s  enantiomers  into  S e c r e t i o n of o t h e r a n t i a r r h y t h m i c drugs  into saliva  has been  s t u d i e d w i t h such agents as (Jaffe  et a l . ,  (Barchowsky et  procainamide (Koup et  1975), disopyramide ( A i t i o al.,  1982)  et a l . ,  and m e x i l e t i n e ( B e c k e t t  Wide i n t e r - and i n t r a - s u b j e c t observed  the  al.,  1975), q u i n i d i n e  1982),  lidocaine  and Chidomere, 1976).  v a r i a t i o n i n saliva/plasma  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  ratios  were  and plasma  c o n c e n t r a t i o n was p o o r . In  the present  study,  the o b j e c t i v e  was not o n l y to  measure  -  saliva after  levels  intravenous  enantiomer interest plasma  of R, S - t o c a i n i d e  164  -  but to measure the i n d i v i d u a l  a d m i n i s t r a t i o n of the racemate and to compare  p r o f i l e w i t h that of plasma and u r i n e .  to determine whether s a l i v a r y  It  s e c r e t i o n of  tocainide  Table 3 1 .  T o c a i n i d e enantiomer l e v e l s i n f u s i o n of 200 mg of  and the s a l i v a  i n the s a l i v a  the racemic drug are  the i n t r a v e n o u s  Throughout the p e r i o d of measurement the isomers  saliva  dose i s  shown i n Table 32 The  being g r e a t e r  f o r the R(-)  one v o l u n t e e r , whose s a l i v a r y pH ranged c o n c e n t r a t i o n of the R(-)  isomer  concentrations,  to 7 . 1 ,  consistently  isomer  (table 33).  high s a l i v a r y  of R(-)  of d a t a i n these v o l u n t e e r s of  to 7.5)  and f o r  appeared  pH.  s l i g h t l y ionised  Tocainide is  u n i t y as  the h i g h e s t  S(+)  saliva  tocainide,  to  1.7.  of s a l i v a / p l a s m a  a weak base (pka = 7.7)  in acidic saliva.  bases u s u a l l y exceed  was o n l y  levels 3-fold  The s a l i v a / p l a s m a  has been r e p o r t e d  procainamide and l i d o c a i n e (Koup et a l . ,  1975;  plasma  Comparison  to suggest a pH dependent  t o c a i n i d e i n t o s a l i v a w i t h the r a t i o  decreasing  the  In another v o l u n t e e r , who had  pH (7.4  t o c a i n i d e was 3.5  In  reached 6 - f o l d h i g h e r  than that of plasma c o n c e n t r a t i o n , w h i l e the d i f f e r e n c e the S(+)  of both  isomer ( T a b l e 3 2 ) .  from 6.9  in saliva  34.  concentrations  were higher than the c o r r e s p o n d i n g plasma  w i t h the d i f f e r e n c e  saliva  shown i n F i g u r e  the s a l i v a r y  are shown  f o l l o w i n g an  to plasma r a t i o s are g i v e n i n Table 33.  p r o f i l e one hour a f t e r  ratio  reflects  concentration.  intravenous  for  the  was a l s o 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 in  isomers  excretion  increasing  and t h e r e f o r e r a t i o of  f o r drugs  is  such weak  such as  Barchowsky et  with  al.,  T a b l e 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  q u a n t i t y of each enantiomer (ng) 50.0  area S(+)T0C  ratios ± S.C.  RSD(%)  R(-•)T0C  RSD(%)  ± 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  3.0566  2.5  3.1059  2.5  1500.0  0.1852  of s a l i v a  Correlation coefficient,  ± 0.0785  r =  0.9998  0.9998  slope  =  1.9987  .2.0446  intercept  = --0.0685  0.0534  The q u a n t i t y of i n t e r n a l s t a n d a r d was 1 u g .  ± 0.0786  Table 32 T o c a i n i d e enantiomers i n the s a l i v a (ug/mL) of h e a l t h y s u b j e c t s f o l l o w i n g an intravenous i n f u s i o n of 200 mg (±) t o c a i n i d e h y d r o c h l o r i d e .  KM  JA  Time (hrs)  pH  0.75  7.1  0.766  1.0  7.2  1.5  RE  SR  pH  (+)T0C  (-)TOC  1.404  7.4  0.935  1.837  -  0.926  1.570  7.4  0.778  1.642  7.0  7.1  1.326  1.874  7.5  0.793  1.468  -  2.0  7.2  1.167  1.475  7.5  0.672  1.212  7.1  0.986  3.0  7.4  1.019  1.379  7.5  0.431  0.618  6.9  5.0  7.1  1.150  1.432  7.3  0.548  0.824  7.0  6.9  0.905  1.107.  7.0  0.529  7.2  (+)T0C  -  pH  (+)T0C  -  (-)TOC  pH  (+)T0C  (-)TOC  pH  (+)TOC  (-)TOC  7.1  1.360  2.337  -  6.9  1.339  2.502  7.4  6.9  1.191  2.130  -  1.638  6.9  1.031  1.724  7.3  0.734  1.077  0.843  1.245  6.9  1.431  2.075  7.1  0.734  1.077  7.0  0.815  1.209  6.9  1.186  1.489  -  0.802  6.9  0.905  1.417  6.9  0.562  0.595  7.6  0.775  1.033  0.113  0.148  7.0  0.926  1.418  7.0  0.400  0.475  7.1  0.665  0.885  1.548  2.122  . -  1.305  -  -  1.964  -  -  10.0  -  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  7.5  0.064  0.063  *32.5 h r s .  -  (-)TOC  CK  0.068  0.056  7.0  0.091  0.032  Table 33 Saliva/plasma  KM Time (hrs)  pH  concentration r a t i o s of t o c a i n i d e enantiomers i n h e a l t h y  JA  (+)T0Cs (+)T0Cp 0.766  pH  (+)TOCs (+)TOCp  (-)TOCs (-)TOCp  1.404  7.4  0.935  1.837  -  7.4  1.473  3.547  6.9  3.086  (-)TOCs (-)TOCp  pH  (+)TOCs (+)TOCp  6.257  7.0  3.005  4.505  7.4  3.117  6.136  -  6.9  2.769  5.199  7.1  2.176  4.015  7.3  2.723  4.724  1.623  6.9  3.788  6.447  6.9  1.821  2.971  7.1  1.835  2.834  1.251  2.119  6.9  3.362  5.350  7.0  2.124  3.754  -  1.658  2.845  6.9  1.956  2.843  6.9  3.067  6.215  7.6  1.992  2.724  7.0  1.492  2.605  7.0  3.379  6.534  7.1  1.837  2.431  7.1  2.702  4.413  7.5  1.653  3.415  6.9  2.00  7.2  2.828  4.339  7.5  1.305  2.616  3.00  7.4  2.280  3.560  7.5  1.053  5.00  7.1  2.721  4.386  7.3  7.00  6.9  2.409  3.628  7.0  -  -  -  2.926  -  -  4.453  -  -  10.00  -  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  7.5  0.771  1.575  7.2  -  -  -  -  (-)TOCs (-)TOCp  -  1.50  -  (+)TOCs (+)TOCp  2.337  -  -  pH  1.360  1.00  -  (-)TOCs (-)TOCp  CK  7.1  7.1  -  (+)TOCs (+)TOCp  (-)TOCs (-)TOCp  0.75  -  RE  SR pH  volunteers.  -  TOCs = t o c a i n i d e i n s a l i v a ; TOCp - t o c a i n i d e i n plasma *32.5 h r s .  -  7.0  3.095  -  7.2  1.658  4.000  -  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 MINUTES  Chromatographic c o n d i t i o n s : Column, C h i r a s i l - V a l ® f u s e d - s i l i c a capillary (50 m x 0.3 mm); i n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; c a r r i e r gas (H2) f l o w , 1 mL/min; make-up gas (N2) f l o w , 30 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 151.7 kPa; c h a r t speed, 0.3 cm/min.  -  1982).  The two f o l d  difference  169  -  i n the r a t i o of the enantiomers  observed i n a l l the f i v e v o l u n t e e r s  cannot be e x p l a i n e d on the b a s i s of  pH dependent i o n i s a t i o n or simple p a s s i v e d i f f u s i o n . c o n c e n t r a t i o n of the R(-) isomer i s period. that  the o p p o s i t e  isomer i n the s a l i v a  as  The h i g h  compared to  of the plasma or u r i n e p r o f i l e at  However, the enantiomer r a t i o  i n the s a l i v a  of s a l i v a p r o f i l e  [R(-)  a l s o cannot be e x p l a i n e d on the b a s i s of excretion into s a l i v a . observation that saliva  as  isomer i s  r a t e of S(+) in saliva result  shorter h a l f - l i f e .  isomer and t h e r e a f t e r  falls  at  a much g r e a t e r  the c o n c e n t r a t i o n of  t i o n of S(+)  >  consistant  It  with  the  from the plasma and  appears that  i n t o the s a l i v a  r a t e than the S(+)  the R(-)  34). R(-)]  at  the R(-) twice  the c o n c e n t r a t i o n of the R(-)  isomer i s  isomer i n most v o l u n t e e r s  The s a l i v a / p l a s m a  approach  (Table  to (S(+)  isomer d i s a p p e a r s f a s t e r  rapidly distributed i n i t i a l l y  to  the known mechanisms of drug  However, the data i s  the R(-)  shown by i t s  > S(+)  S(+)  the same time  tends  of plasma and u r i n e p r o f i l e by 48 hours ( F i g u r e 35) This reversal  as  after  less  isomer.  the isomer  As a  than the c o n c e n t r a -  24 h o u r s .  c o n c e n t r a t i o n of a b a s i c  drug can be p r e d i c t e d  a c c o r d i n g to the e q u a t i o n :  s/p  =  1 + 10  (pka -  P  HS)  (pka -  P  Hp)  x f  p  /f  s  1 + 10  where p k a i s plasma  the p k a of the b a s i c drug that  to s a l i v a ,  pHp is  the pH of  is  the plasma,  transported pHg is  from  the pH of  the  -  170 -  FIGURE 35  LOG FOR  PLASMA R(-)-  AND AND  SALIVA CONCENTRATIONS S(+)-  TOCAINIDE  vs  TIME  ENANTIOMERS  3 CO  z  o  t< cc tz LU  o z o o < >  < CO  S(+)tocainide in s a l i v a  o z <  S(+)tocainide in p l a s m a R(-)tocainide in s a l i v a  <  R(-)tocainide in p l a s m a  CO  < o o  20 TIME  30  40  (HOURS)  50  Table 34 Enantiomer r a t i o , (+)T0C/(-)T0C, i n plasma, urine and s a l i v a f o l l o w i n g intravenous of 200 mg (±) t o c a i n i d e hydrochloride to h e a l t h y male v o l u n t e e r s .  JA  KM Time (hrs)  P*  0.33  1.04  0.75 1.00  U*  S*  P  -  1.13  1.12  -  0.54  -  0.98  RE  SR  0  S  P  -  1.09  1.03  -  0.51  0.59  1.14  1.03  infusion  U  S  -  1.00  1.07  -  0.58  0.73  1.08  0.99  U  S  1.07  -  -  0.47  1.09  0.97  P  CK  1.03  U  S  0.98  -  -  0.53  1.00  0.96  0.68  P  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  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 = S a l i v a  '  0.80  -  -  -  -  -  saliva  at  -  the time of s e c r e t i o n of the d r u g , fp i s  drug unbound i n plasma and f s is  172  is  the f r a c t i o n of  the f r a c t i o n unbound i n s a l i v a  the  which  u s u a l l y taken as u n i t y . Tocainide is  E l v i n et a l . , in 0.8  (1982) have shown that  the serum of to 0.9  not bound to plasma p r o t e i n s  glycoprotein).  the range of 4-12  ug/mL.  tocainide  to 0.96  and from  (who had h i g h c o n c e n t r a t i o n s of  of t o t a l  from 85  Using f p  = 0.9,  p k a = 7.7  and pHp 7.4  (table 35).  Although there was a marked i n t e r s u b j e c t  for  S(+)  tocainide;  for  the R(-)  isomer,  in  r a t i o of t o c a i n i d e has been The observed  were always h i g h e r than p r e d i c t e d and the c o r r e l a t i o n was  ratios  poor.  v a r i a b i l i t y between the  (1.406 to 2.603 w i t h a mean of 2.068 ± 0.508 (SEM) 2.914  to 4.374 with a mean of 3.683 ± 0.760 (SEM)  the c o r r e l a t i o n between the s a l i v a  c o n c e n t r a t i o n i n the same i n d i v i d u a l was good, 0.884 to 0.986 f o r  the R(-)  and plasma  ( r = 0.910  isomer)  (Fig.  to 0.987  S(+)  isomer;  6.1  T o c a i n i d e Enantiomers i n t h e S a l i v a o f a P a t i e n t w i t h R e n a l  for  36).  Dysfunction The measured  salivary  to  to be independent of serum c o n c e n t r a t i o n w i t h i n  and compared with the r a t i o observed  saliva/plasma  a^acid  with the above study were  unbound t o c a i n i d e ranged  the above e q u a t i o n , the s a l i v a / p l a s m a  ratios  extent.  (1982) u s i n g serum samples from 5 h e a l t h y  The percentage  90% and b i n d i n g appeared  predicted  ranged from 0.78  Binding data c o n s i s t e n t  r e p o r t e d by Sedman et a l . volunteers.  the unbound f r a c t i o n of  10 h e a l t h y v o l u n t e e r s  i n 4 trauma p a t i e n t s  to any g r e a t  c o n c e n t r a t i o n s of S(+)  i n the s a l i v a of a p a t i e n t  and R(-)  with t o t a l  t o c a i n i d e were  renal failure  (who was  -  173 -  T a b l e 35 Comparison of observed and p r e d i c t e d s a l i v a / p l a s m a r a t i o s of t o c a i n i d e i n one of the h e a l t h y v o l u n t e e r s (KM)  P r e d i c t e d s/p Time (hrs)  pH  0.75  7.1  1.50  Observed s/p r a t i o —  S(+)  ratio  Uncorrected  Corrected  1.458  1.662  1.496  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 B E T W E E N SALIVA AND PLASMA L E V E L S OF TOCAINIDE ENANTIOMERS FOLLOWING INTRAVENOUS INFUSION O F R.S-TOCAINIDE  !.0  E cn  1.5  U z  o o  S(+) Tocainide  < >  <  r=0.9289  1.0  CO  0.5  0.1  "02"  "03"  0.4  P L A S M A C O N C . ug/mL  0.5  -  undergoing h e m o d i a l y s i s a d m i n i s t r a t i o n of  three times  the racemic d r u g .  the same time p e r i o d s  as  for s a l i v a .  the isomers  are g i v e n i n Table 36.  the isomers  as w e l l  patient  as  Plasma l e v e l s  were a l s o measured  The s a l i v a / p l a s m a The s a l i v a r y  the s a l i v a / p l a s m a  ratios It  r a t i o of t h i s weakly b a s i c  governed by pH of the s a l i v a throughout  -  a week) f o l l o w i n g i n t r a v e n o u s  compared to h e a l t h y v o l u n t e e r s .  the s a l i v a / p l a s m a  D.  175  (24  of both  concentrations  of both  were very h i g h i n t h i s  appears from t h i s data drug ( p k a = 7.7)  (pH was c o n s i s t e n t l y h i g h ,  the p e r i o d of measurement  ratios  at  8.0  is  -  that  not  8.4  hrs).  Analysis o f Tocainide Metabolites i n the Urine T o c a i n i d e has been r e p o r t e d to be m e t a b o l i z e d i n man by a n o v e l  pathway to a c a r b a m i n i c a c i d f o l l o w e d by c o n j u g a t i o n w i t h g l u c u r o n i c acid  ( E l v i n et  al.,  1980b).  T h i s g l u c u r o n i d e , u n l i k e any p r e v i o u s l y r e p o r t e d i s cyclisation 1980b).  thought to undergo  at pH 13 to a h y d a n t o i n - t y p e compound ( E l v i n et  TOCG belongs  carboxy group i s non-steroidal  to the group of e s t e r - t y p e  conjugated w i t h g l u c u r o n i c a c i d .  a n t i i n f l a m m a t o r y drugs  a c i d and k e t o p r o f e n are examples  such as  al.,  g l u c u r o n i d e s where a Conjugates  of  various  indomethacin, s a l i c y l i c  of e s t e r - g l u c u r o n i d e s .  The e s t e r  -  176 -  T a b l e 36 T o c a i n i d e enantiomers :i n the s a l i v a of a p a t i e n t w i t h r e n a l d y s f u n c t i o n and s a l i v a / p l asma r a t i o s •  •  Time (hrs)  pH  1.0  (+)T0C g  (-)T0C8  (+)T0C  (-)TOC  (+)T0C (Ug/mL)  (-)TOC (ug/mL)  ratio  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  T0Cs = tocainide i n s a l i v a TOCL, = t o c a i n i d e i n plasma  P  P  -  glucuronides  i n g e n e r a l are u n s t a b l e  d u r i n g s t o r a g e i n the f r e e z e r ketoprofen glucuronide is stored  is  al.,  glucuronides  is  and a l s o  For example,  known to h y d r o l y s e s l o w l y when the u r i n e  or 4 .  1980a).  1980).  is  Another problem w i t h e s t e r  Recent r e p o r t s  and Doust,  The aglycone  can migrate  suggest such m i g r a t i o n et  al.,  1982;  Only one of the f o u r  for  Upton et possible  h y d r o l y s e d by the enzyme,*,, 3 - g l u c u r o n i d a s e .  a l l glucuronides  3-glucuronidase  solutions  1980).  probenecid and k e t o p r o f e n (Hasegawa  1980a; Eggers  that  in alkaline  intramolecular acyl migration.  from p o s i t i o n 1 to 2,3 zomepirac,  -  (Upton et a l . ,  at - 1 5 ° C (Upton et a l . ,  glucuronides  177  The  fact  are not h y d r o l y s e d by the commonly used  must be c o n s i d e r e d w h i l e d e v e l o p i n g assay methods  for  glucuronides. T o c a i n i d e carbamoyl g l u c u r o n i d e i s the extent hours  of about  ( E l v i n et  al.,  1980a).  glucuronides.  glucuronidation is a substantial is  Since t o c a i n i d e has  It  interest  to e s t a b l i s h  an  1.  renal dysfunction a l t e r  analytical  one c h i r a l  72  center,  for c h i r a l excreted  whether  that  compounds.  process.  Since  as g l u c u r o n i d e s ,  Such a study  technique to measure the e n a n t i o m e r i c  states  necessitates  glucuronides,.  Gas Chromatographic A n a l y s i s o f G l u c u r o n i d e s G l u c u r o n i d e s have been c h a r a c t e r i z e d by examination of  aglycone r e l e a s e d  it  stereoselective  drug and a l s o whether d i s e a s e this  to  i n the f o r m a t i o n of two  generally recognized  a stereoselectiveprocess  g l u c u r o n i d a t i o n occurs with t h i s such as  is  result  f r a c t i o n of t o c a i n i d e dose i s  of c o n s i d e r a b l e  i n the human u r i n e  30% of the a d m i n i s t e r e d dose w i t h i n the f i r s t  conjugation with glucuronic acid w i l l diastereomeric  excreted  by a c i d or enzymatic h y d r o l y s i s .  the  Widespread concern  -  exists about  about  -  the s e l e c t i v i t y of both enzymic or chemical h y d r o l y s i s and  the wide v a r i a t i o n of t h e i r r a t e s of h y d r o l y s i s and the o p t i m a l  conditions with d i f f e r e n t be a l t e r e d  substrates.  1982).  Thus h y d r o l y s i s i s  and the r a t i o n a l e  glucuronides.  In recent  3_glucuronidase,  for analysis techniques  of i n t a c t g l u c u r o n i d e s . that  Frequent procedures trifluoroacetylation Thompson et a l . ,  thermally stable  are m e t h y l a t i o n ,  1973,  1975)  the m e t h y l a t i o n of g l u c u r o n i d e s chromatography/mass  or of  intact  have been  thermal is  therefore  described  In a s e r i e s of  papers,  a one-step procedure  169,  141,  Many c h a r a c t e r i s t i c  glucuronides during  101 and 75 can be observed  of  ion  fragments 232,  However,  the e s t e r - t y p e are known to be p a r t i a l l y h y d r o l y s e d  Decarbamylation takes p l a c e  2.  i n the mass s p e c t r a .  the process of p e r m e t h y l a t i o n (Thompson et a l . ,  (Lynn et a l . ,  for  by gas  d e r i v e d from the permethylated g l u c u r o n i c a c i d p o r t i o n such as m/z 201,  al.,  derivatives.  and t h e i r a n a l y s i s  spectrometry.  et  acetylation,  and t r i m e t h y l s i l y l a t i o n .  (1972,  It  may  Glucuronides  u s u a l l y undergo  i n GC systems p r i o r to v o l a t i l i z a t i o n .  n e c e s s a r y to prepare v o l a t i l e ,  (Hasegawa  quantitatively  clearly exists  p o l a r p o l y f u n c t i o n a l molecules  degradation  either  years chromatographic  to the c h a r a c t e r i s a t i o n  of the aglycone may  to the same extent  not r e l i a b l e  qualitatively,  applied  The s t r u c t u r e  by chemical h y d r o l y s i s and the enzyme,  not h y d r o l y s e i s o m e r i c g l u c u r o n i d e s  are  178  1973).  i n the case of carbamazepine  glucuronide  1978).  Liquid Chromatographic Analysis of Glucuronides Liquid  chromatography o f f e r s  chromatography f o r  an a t t r a c t i v e  alternative  to  gas  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 p o l a r  - 179 -  metabolites assay i s  such as  glucuronides.  conducted at  No d e r i v a t i s a t i o n  room temperature.  moiety does n o t c o n t r i b u t e  is  r e q u i r e d and  the  Although the g l u c u r o n i c a c i d  to u l t r a v i o l e t a b s o r p t i o n , many aglycones  do  have some chromophoric g r o u p s .  Separation  of D i a s t e r e o m e r i c G l u c u r o n i d e s  The f o r m a t i o n of a g l u c u r o n i d e from a c h i r a l molecule i s to d e r i v a t i s a t i o n diastereomers. properties,  i n GC w i t h o p t i c a l l y a c t i v e  Since diastereomers  it  is  possible  reagents  have d i f f e r e n c e s  to r e s o l v e  them by l i q u i d  buffer  glucuronides  resolved  (Hermansson e t a l . ,  reported  for  phosphate  of hydantoin have been  A s i m i l a r r e s o l u t i o n a l s o has  as  the mobile phase (Thompson e t a l . ,  been  1981).  I s o l a t i o n o f G l u c u r o n i d e s from P r i n e S o l v e n t e x t r a c t i o n methods are not very s u i t a b l e  i s o l a t i o n of g l u c u r o n i c a c i d conjugates from b i o l o g i c a l Activated  charcoal is  conjugates.  sometimes  used  to adsorb  A more commonly used adsorbent  aqueous s o l u t i o n s  i s XAD-2  solutions.  the  materials.  glucuronic acid polar metabolites  from  n o n i o n i c , s t y r e n e - d i v i n y l benzene  co-polymer h a v i n g a l a r g e s u r f a c e a r e a . the a d s o r p t i o n of water  for  for  resin.  A m b e r l i t e XAD-2 r e s i n i s  is  in  p r o p r a n o l o l g l u c u r o n i d e s employing an RP-18 column and  methanol/ammonium phosphate  3.  1982).  physical  chromatography.  and 8% a c e t o n i t r i l e  two d i a s t e r e o m e r i c  to form  in their  U s i n g a r e v e r s e - p h a s e column (Cj^) (pH 7.5)  similar  soluble  Its  most s i g n i f i c a n t  organic species  XAD a d s o r p t i o n does not separate  property  from aqueous  the parent drug from  its  -  metabolites urine  but i s  a useful  concentration step.  the drug and m e t a b o l i t e s  organic solvents. unlikely  It  is  180 -  From a l a r g e volume of  can be obtained i n a c o n c e n t r a t e d  applicable  to cause any u n d e s i r a b l e  to a l l  form i n  types of g l u c u r o n i d e s and  structural  a l t e r a t i o n of  is  the  conjugates. Fujimoto and Wang (1970) were two of describe  a simple procedure for  from u r i n e with XAD-2 r e s i n . pigments were a l s o adsorbed fraction  as  the drugs  Their  e a r l y work showed that u r i n a r y  and m e t a b o l i t e s .  i n the same  Thus, the f r a c t i o n w i t h  the  to the f r a c t i o n c o n t a i n i n g  and t h e i r m e t a b o l i t e s .  Bradlaw (1968)  and simple i s o l a t i o n procedure u s i n g n e u t r a l XAD-2  the e x t r a c t i o n of s t e r o i d g l u c u r o n i d e s  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 f o r glucuronide involved  to  analgesics  on to the r e s i n and were e l u t e d  the g r e a t e s t amount of drugs  r e s i n for  investigators  the e x t r a c t i o n of n a r c o t i c  g r e a t e s t c o l o u r i n t e n s i t y a l s o corresponded  reported a successful  the f i r s t  four major steps ( F i g u r e  37):  1.  A d s o r p t i o n on XAD-2  2.  T h i n l a y e r chromatography on a p r e p a r a t i v e reverse-phase  tocainide  resin. scale using  p l a t e s and i s o l a t i o n of bands c o r r e s p o n d i n g to  glucuronide. 3.  P e r m e t h y l a t i o n and a n a l y s i s  by gas  chromatography/mass  spectrometry. 4.  Direct analysis  by l i q u i d  chroraatography/mass  spectrometry.  - 18-1 -  FIGURE 37  ISOLATION  OF T O C A I N I D E G L U C U R O N I D E S  FROM  HUMAN U R I N E  urine  I elute with H 0 y e l u t e w i t h Me OH MEOH  EXT.'  vac. evaporate, f r e e z e d r y GC, FREEZE  NaOH SAMPLE  HYDANTOIN  IN  >  GC/MS  I TLC MEOH: 60  H 0 2  WITH  : 40  LC DERIVATIVE  ECD<-  BANDS  NAPHTHORESORCINOL  SPRAY  REAGENT  I HYDANTOIN  GC,  VISUALIZE  FID  GC/MS  LC  DERIVATIVE  PH=13  , MEOH  GC,  FID  DRIED  NaOH pH=l:  ISOLATE  BANDS  CORRESPONDING GLUCURONIDES  V  6-glucouronidase pH=5. TOCAINIDE ISOMERS  TO  LC/MS H 0 2  90  Hcl  100° PERMETHYLATION DMSO,  r  CH I  GC/MS  3  :  CH CN 3  : 10  - 182 -  A.  M i c r o p o r e LCMS o f T o c a i n i d e The  Glucuronide  p o s i t i v e i o n mass spectrum o b t a i n e d  t i o n mode i s shown i n F i g u r e 38.  i n the chemical  ionisa-  The i o n a t m/z 236 corresponds  to a  ft ft  HCCHNHCOH  s t r u c t u r e which i s the aglycone  m/z 236  o f t o c a i n i d e carbamoyl g l u c u r o n i d e .  The  i o n a tra/z219 was formed e i t h e r by l o s s of an -OH group l e a d i n g to the acylium  Ion, or by c y c l i s a t i o n  M/Z  to a hydantoin  of 'acylium'  i o n s from e s t e r s ( B u d z i k i e w i c z e t a l . , 1967) and 1976) have been r e p o r t e d .  i o n s p e c t r a a l s o contained  m/z 165, m/z 162 (base  219  Hydantoin  e s t e r - g l u c u r o n i d e s (Baake e t a l . , positive  K/Z  219  Acylium ion  Formation  s t r u c t u r e as shown below:  The  fragments such as m/z 197, m/z 181,  peak) and m/z 153 but a s t r u c t u r e f o r these i o n s  -  183  -  FIGURE <38;  '  MICROBORE LCMS OF TOCAINIDE GLUCURONIDES  i6a  236  528^  O H Q H H O  |  N-C-C-N-C—^  1  I  CH, 36&\  CH,  IX  ICI- O H  OH OH  N carboxy tocaimda glucuronic)*  MW • 412  236  280-1 219 208^  120H 181  X  4B-  165  153  197 /  160  180  200  220  -  c o u l d not be r a t i o n a l i z e d . fragment, negative  m/z 119.  The n e g a t i v e  conditions.  In a d d i t i o n to m/z 119,  Siegmund (1982) have shown that at a source  119 and that Since  temperature  the p a t t e r n of  the s p e c t r a  was a l s o  this  of 2 2 0 ° C was  the  fragmentation  C a i r n s and  was  temperature  it  is  believed  that both of  from the g l u c u r o n i c a c i d m o i e t y .  added more d i r e c t e v i d e n c e f o r  glucuronic  sensitive.  of p - n i t r o p h e n o l g l u c u r o n i d e ( F i g u r e 39)  i o n mode),  also  the dehydrated p r o d u c t , m/z  m/z 119 (-ve  5.  spectra  the base peak obtained f o r  c o n t a i n e d m/z 162 i o n (+ve  carbamoyl  the base peak i n  to p - n i t r o p h e n o l .  g l u c u r o n i d e ( F i g u r e 38)  derived  c o n t a i n e d only one  of p - n i t r o p h e n o l g l u c u r o n i d e o b t a i n e d under  c o n t a i n e d m/z 139 which corresponds  acid  ion spectra  The same i o n (m/z 119)  ion spectra  identical  184-  and  tocainide  i o n mode), as w e l l these ions were  T h u s , a microbore LCMS  the proposed  as  structure  of  study  tocainide  glucuronide.  I d e n t i f i c a t i o n o f Band l b as T o c a i n i d e 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  h y d a n t o i n - t y p e compound by a d j u s t i n g et a l . ,  1980).  derived  from a g l u c u r o n i d e of  derivative  F i g u r e 40 shows  the pH of  fragmentation  proved by comparison of  patterns  mass spectrometry  of  ( F i g u r e 41 and 4 2 ) .  obtained by c a p i l l a r y  the r e f e r e n c e  (Elvin  the h y d a n t o i n  along w i t h standard  s u p p l i e d by A s t r a P h a r m a c e u t i c a l s .  h y d a n t o i n was f u r t h e r  the s o l u t i o n to 13  the chromatogram of  tocainide  to a  hydantoin  The i d e n t i t y of  the  the r e t e n t i o n times and column gas  chromatography/  substance and the i s o l a t e d  material  - 185;..^  FIGURE 3 9 MICROBORE LCMS OF p-NITROPHENOL GLUCURONIDE  157  lseia-  COOH  1000-  MW 315 162 5BB  181  .155 .L  198  165  175  185  19S  - 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 c o n d i t i o n s : I n j e c t i o n temperature, 2 4 0 ° C ; d e t e c t o r (FID) temperature, 3 0 0 ° C ; oven temperature, 1 9 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; make-up gas (He) f l o w , 60 mL/min; s p l i t vent f l o w , 30 mL/min; column i n l e t p r e s s u r e , 55.1 kPa; c h a r t speed, 0.3 cm/min.  -  188  -.  FIGURE .42 "  CI M A S S S P E C T R A O F A HYDANTOIN DERIVATIVE OBTAINED FROM A GLUCURONIDE O F TOCAINIDE  Scan 8 . 8 5 min.  219 (M+1)  108  CH.  0  CH,  O  148 >  CH  MW 218  203  iaa  iza  i4-a  i6a  tea  2ee  220  z*a  zee  ZBB  - 189 -  The  fraction labelled  h y d r o l y s i s with  as band I b  ft-glucuronidase  showed that more of  the R(-)  there i s more of R(-)  a l s o gave t o c a i n i d e  or 1 N HC1 ( F i g . 4 3 ) .  isomer i s  generated  after  This  chromatogram  by h y d r o l y s i s and  t o c a i n i d e g l u c u r o n i d e i n u r i n e than S(+)  that  tocainide  glucuronide. T h i s was f u r t h e r u r i n e as w e l l hydrolysis,  as  supported  band 1^ w i t h ^ - g l u c u r o n i d a s e  the q u a n t i t y of R(-)  p r o f i l e of unhydrolysed u r i n e . acid  6.  by a more s e l e c t i v e  h y d r o l y s i s of  enzyme.  After  t o c a i n i d e was i n c r e a s e d Band I a  d i d not g i v e  the  compared  tocainide  to  the  after  or enzyme h y d r o l y s i s .  Gas Chromatographic/Mass S p e c t r o m e t r l c A n a l y s i s o f P e r m e t h y l a t e d Glucuronides Fractions  isolated  methanolic extract  by p r e p a r a t i v e  TLC as w e l l  of u r i n e were, permethylated  as  for gas  the f r e e z e  dried  chromatographic  analysis.  6.1  CI and E I Mass S p e c t r a o f Permethylated The  presented  CI mass spectrum of permethylated g l u c u r o n i c a c i d here as a r e f e r e n c e  There was no m o l e c u l a r i o n present at m/z 293 which corresponds reactant  gas).  group ( M - O C H 3 ) .  (Figure 44,  at m/z 264.  to M + C2H.5  +  i o n m/z 233  45 and Table  37).  The h i g h e s t mass i o n was  (probable when methane i s  The i o n , m/z 233 corresponds The fragment  is  to a i d i n the i n t e r p r e t a t i o n of  permethylated g l u c u r o n i d e mass s p e c t r a  as  Glucuronic Acid  used  to l o s s of a methoxy  then l o s e s MeOH  successively  -  190 -  FIGURE -43  CHROMATOGRAM OF HEPTAFLUOROBUTYRYL DERIVATIVES OF TOCAINIDE ENANTIOMERS OBTAINED BY ACID-HYDROLYSIS OF BAND Iv,  1.  S (+)  tocainide  2.  R (-)  tocainide  Chromatographic c o n d i t i o n s : 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 ; d e t e c t o r (ECD) temperature, 3 5 0 ° C ; oven temperature, 2 0 0 ° C ; c a r r i e r gas (He) f l o w , 1 mL/min; s p l i t vent f l o w , 30 mL/min; make-up gas (N2) f l o w , 30 mL/min; c h a r t speed, 0.3 cm/min.  -  191  -  FIGURE 44 GC/MS/CI OF PERMETHYLATED GLUCURONIC ACID COLUMN:  SE-30 FUSED-SILICA CAPILLARY  8 8  6 0  20  «e  1  20  160  200  240  280  328  360  -  192  -  FIGURE 45  FRAGMENTATION  PATTERN  CH 0 = CHOCH 3  M/Z  OF  PERMETHYLATED  GLUCURONIC  CH 0=CHCH=CHOCH  3  3  M/Z  75  COOCH,  101  /  o-  CH36 |  OCH  3  OCH, MW  COOCH  / " y/-0CH  264  y-COOCHa  3  OCH  CH,0  3  OCH3 M/Z  M/Z  233  - CH0H 201  3  M/Z  CH0H 169  173  CH OH  3  M/Z  205  CH OH  3  M/Z  0CH OCH3  3  M/Z  141  3  3  ACID  - 193 -  to g i v e m/z 201 by  cleavage of  and m/z 169.  The i o n s , m/z 101  the pyranoside r i n g .  The m/z 201  intense  i o n i n the CI mass spectrum ( T a b l e 37).  101 was  the most i n t e n s e  ions  such as m/z 141,  spectrum.  The f a c t  and m/z 75 were formed i o n was  On the other hand, m/z  i o n i n the EI mass spectrum.  88 and 75 were a l s o i n c r e a s e d  that ions such as m/z 205,  the most  173  o n l y under e l e c t r o n impact i o n i s a t i o n suggested  Intensities  of  i n the EI mass and 141 were formed  that removal of -COOCH3  group may be a prominent s t e p i n v o l v e d under these c o n d i t i o n s .  6.2  C I Mass S p e c t r a o f Permethylated As  shown i n the CI mass spectrum ( F i g u r e 46 and T a b l e 3 8)  M + 1 i o n at m/z 372 mass i o n at m/z 400 to M-OCH3. acid  p-nitrophenol glucuronide  was  the most i n t e n s e  i n the s p e c t r a .  The h i g h e s t  corresponded to M + C2H5 + and m/z 340  corresponded  Fragments c h a r a c t e r i s t i c  p o r t i o n of  the  of  the permethylated g l u c u r o n i c  the molecule such as m/z 233,  were a l s o seen i n the spectrum.  m/z 201,  The i o n , m/z 139 was  m/z 169, that of  m/z  145,  the  a g l y c o n e , p - n i t r o p h e n o l which was not e v i d e n t i n the EI mass spectrum. Additional  fragments  such as m/z 173,  141,  88 and 75 were a l s o formed  under e l e c t r o n impact c o n d i t i o n s from the g l u c u r o n i c a c i d m o i e t y .  6.3  GCMS o f P e r m e t h y l a t e d T o c a i n i d e G l u c u r o n i d e s U s i n g an SE-30 F u s e d - s i l i c a C a p i l l a r y Column T o c a i n i d e g l u c u r o n i d e s (band 1^)  thin-layer  isolated  by p r e p a r a t i v e  chromatography were permethylated and analysed  chromatography/mass  by gas  spectrometry u s i n g an SE-30 f u s e d - s i l i c a  capillary  - 194 -  T a b l e 37 R e l a t i v e i n t e n s i t i e s of fragment i o n s of permethylated g l u c u r o n i c a c i d i n the CI and EI mass s p e c t r a .  m/z  relative  intensity % CI  relative  intensity % EI  293  9  -  263  3  -  233  19  -  205  -  2  201  100  1  173  -  2  169  50  5  145  3  4  141  -  101  11  88 75  8  2 100 29 36  - 195 -  FIGURE 46 .  QC/MS/CI  OF  PERMETHYLATED  p-MITROPHENOL  column:SE-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  ise  see  «ee  OLUCURONIDE  injection,methane  reagent  gas  - 196 -  Table .38 R e l a t i v e i n t e n s i t i e s of fragment ions of permethylated p - n i t r o p h e n o l g l u c u r o n i d e i n the CI and EI mass s p e c t r a .  m/z  relative  intensity % CI  relative  intensity % EI  400  16  -  372  100  -  340  3  -  308  9  -  240  24  -  3  9  201  29  100  177  24  -  173  -  232'  7  169  4  14  145  10  3  141  -  139  4  27  -  101  -  37  88  -  13  75  -  30  - 197 -  column (25 m x 0.2  mm).  The permethylated  sample was i n j e c t e d  onto  the  column by the s p l i t l e s s mode and the oven was programmed from 5 0 ° to 140° was  at 3 0 ° / m i n operated  and from 1 4 0 ° to 2 4 0 °  the E l mass s p e c t r a of  the  of  the  total  The mass  ion current p r o f i l e  the peak e l u t i n g at  three compounds e l u t i n g at  c o n t a i n e d many of (Table 39).  10°/min.  spectrometer  i n the EI mode.  F i g u r e 47A shows  spectra  at  the same fragment  A l l the  18.9 m i n u t e s . 18.6,  18.9  and 19.4  the c h r a c t e r i s t i c  141,  101, 88 and 75)  reported  for  permethylated g l u c u r o n i d e s of l a b i l e the EI s p e c t r a ) . fragments  The  i o n at  for  T a b l e 39 shows  the three  tocainide  fragments  of v a r i o u s  the  seen i n Ion  compounds.  abundance i n a l l  was probably d e r i v e d from the permethylated  glucuronide since  of  i o n s of  compounds are not always  (m/z 277)which was found i n c o n s i d e r a b l e  three s p e c t r a ,  of a  but none showed  (molecular  the i n t e n s i t i t e s  mins.  intensity  a m o l e c u l a r i o n which c o u l d be c o r r e l a t e d w i t h the s t r u c t u r e glucuronide metabolites  is  The mass  ions but w i t h v a r y i n g  three had most of  permethylated g l u c u r o n i d e (m/z 201,  and F i g . 47B  such fragmentation  is  known to occur f o r  ester esters  the  -  198 -  FIGURE 47  GC/MS  OF PERMETHYLATED OF  GLUCURONIDES  TOCAINIDE column S E 3 0 f u s e d •lllca(is-m>ozs) splitless  Injection s , m  o v e n temp.  4  e  ia  105  16  2B  50  24  28  5 mm  32  36  43  10s  98  _277  CH,.H 0 H H I II I I " -C-N yO/rN~c-(f-I N-(--Ov CH,  156  44  18 93 m i n .  EI Mode  100  80  ^2G0'  2 3 a * 201,141,101,75 0 II  C-OCH,  0 1 \  C M > 0  /\  J *0CH, OCH,  6B 2B1 72  141  277  40  12J 20  ITl  +4.  232  _il_ iaa  20B  3aa  334  i 430  sea  - 199 -  Table 39 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 l u c u r o n i d e s of t o c a i n i d e e x t r a c t e d 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)  R e t e n t i o n 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)  R e t e n t i o n 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  ( B u d z i k i e w i c z e t a l . , 1967). spectrum (ra.w. of  The  -  i o n at ra/z 192  t o c a i n i d e = 192).  was  not seen i n the  However, there was  evidence  f o r the  presence of the i o n :  m/z  105  which was  the most i n t e n s e (100%) f o r the peak e l u t i n g at 18.9  The  i o n fragments, m/z  three  156,  157  and 98 were present i n a l l the  s p e c t r a but a s t r u c t u r e c o u l d not be d e f i n i t e l y The  total  i o n c u r r e n t p r o f i l e ( F i g . 51a) taken  assigned.  a l s o i n d i c a t e d that e x t e n s i v e  degradation  had  glucuronide  or d u r i n g chromatographic a n a l y s i s .  p l a c e d u r i n g d e r i v a t i s a t i o n of the e s t e r  mins showed the f o l l o w i n g i o n fragments:  75,  a l l of which are c h a r a c t e r i s t i c of permethylated at l e a s t p a r t i a l d e c o n j u g a t i o n  spectrum of 176,  148,  the peak e l u t i n g at 6.7  105  and  77.  The  proposed  m/z  The  4.8  suggesting  mins.  compound e l u t i n g  201,  141,  101,  88  glucuronic  of the g l u c u r o n i d e .  and  acid, The  mins showed fragments such as s t r u c t u r e of these  at  mass m/z  ions are:  HCCH  m/z  176  m/z  148  m/z  105  m/z  77  -  Therefore, The  201  -  t h i s peak must be t o c a i n i d e or d e r i v e d most i n t e n s e  from  tocainide.  Ion (100%) i n the mass s p e c t r a of compounds  e l u t i n g a t 8.4, 9.0, 9.2, 9.5, 10.1,  and 10.2 minutes was m/z 135 which  corresponds t o :  m/z 135  This  fragment i o n c o u l d  nitrogen  6.4  raethylation  by"raethyliodide i n the presence of dimsylsodiura.  there was no other during  have been formed by the  evidence  of the amide However,  to suggest t h a t such an a r t i f a c t  arose  the d e r i v a t i s a t i o n p r o c e s s .  GCMS o f P e r m e t h y l a t e d T o c a i n i d e G l u c u r o n i d e s  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 c l o s e s i m i l a r i t y  of the mass s p e c t r a 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 f u r t h e r 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 t h i s sample was made u s i n g the Chirasil-Val® f u s e d - s i l i c a column.  F i g u r e 48A shows the t o t a l i o n  c u r r e n t p r o f i l e of the permethylated f r a c t i o n and the E I mass spectrum of the peak e l u t i n g a t 7.91 minutes i s shown i n F i g u r e 48B. yeilded  This  peak  abundant mass s p e c t r a l evidence f o r the presence of g l u c u r o n i d e  c o n j u g a t e of t o c a i n i d e s t r u c t u r e assignments f o r those fragments a t m/z  -  202 -  FIGURE 48  QC/MS/EI OF P E R M E T H Y L A T E D  GLUCURONIDE  OF TOCAINIDE Column: C h i r a s i l - V a l ( 1 5mx0.31 mm)  "Z.  oo ^ o o 6 7 -  6.  12  18  14-  16  2 en  B 121  176 192  \ Y  CH \ 3  277  7 . 9 1 min.  COOCH,  NHCoicHNHiCOO  !|  :  :  CH  1  CH, ,05  iCH  1 4 8  3  3  ^232  MW 468 232  101 141 7S  116 169  »5  45  156  U  29  56  145  9*  233 192  277  34B  X 40  TO  13°  i6«>  200  240  asa  32e  -  192,  176,  148,  121,  t o c a i n i d e p o r t i o n of 101,  105,  203  -  90 and 77 could be c o r r e l a t e d w i t h  the m o l e c u l e , w h i l e fragments  and 75 c o u l d be c o r r e l a t e d w i t h those  the  at m/z 233,  shown p r e v i o u s l y to  201,  169,  arise  from a permethylated sample of g l u c u r o n i c a c i d . Those peaks e l u t i n g at 9 . 2 1 ,  9.80  and 11.49 minutes  e x h i b i t e d a number of ions which were c h a r a c t e r i s t i c glucuronide. hence probable  However, evidence f o r structures  were not  the  also  of a permethylated  t o c a i n i d e moiety was weak and  assigned.  - 204 -  SUMMARY AND CONCLUSIONS  A new g a s - l i q u i d low l e v e l s silica  of ( ± ) t o c a i n i d e  assay method f o r measurement  in biological  fluids  c a p i l l a r y column and e l e c t r o n capture  developed. for  chromatographic  The a p p l i c a b i l i t y of  pharmacokinetic  humans.  studies  The method has a l l  i n e r t and f l e x i b l e  d e t e c t i o n has  technique has  i n small laboratory  capillary  animals  by e l e c t r o n capture  d i r e c t r e s o l u t i o n of r e a d i l y enantiomers  enantiomeric  prepared  tocainide  and e l e c t r o n capture  advantages compared  for  the f i r s t  selective  is  based upon the  detection.  of  of  D e t e r m i n a t i o n of  u r i n e and s a l i v a  by  this  to assays based on r e s o l u t i o n of  the pharmacokinetics  following administration  of  detection.  the d i a s t e r e o m e r s on c o n v e n t i o n a l packed columns. technique d e v e l o p e d ,  and a l s o i n  heptafluorobutyryl derivatives  composition i n human plasma,  method has d e f i n i t e  demonstrated  combined w i t h  The s t e r e o s e l e c t i v e assay method developed  tocainide  been  columns and the h i g h  i n h e r e n t w i t h the system,  and s e n s i t i v e d e t e c t i o n  been  fused-  the advantages a s s o c i a t e d with the use  fused-silica  resolution capability  this  employing a  of  Employing the new  of i n d i v i d u a l enantiomers  the racemic drug have been  of  studied  time i n h e a l t h y s u b j e c t s and i n a p a t i e n t w i t h r e n a l  dysfunction. The s t e r e o s e l e c t i v e method was found measurement revealed  of  that  enantiomers  tocainide there i s  enantiomers  in saliva  enantioselectivity  i n t o human s a l i v a  to be a p p l i c a b l e  and that  as w e l l .  This  i n the e x c r e t i o n of  the enantiomeric  to study  has  tocainide  c o m p o s i t i o n of  -  the s a l i v a  is  observations drug i n t o saliva  different are  205  -  from that of plasma  i n d i c a t i v e of a s p e c i a l  or pKa of  the c l e a r a n c e dialysis  that  2.5-fold is  on the b a s i s of pH of  the h a l f - l i v e s  compared  of both  to h a l f - l i v e s  decreased by 40%.  enantiomers  i n healthy su b j e c ts, while  The f r a c t i o n of  glucuronides evidence  glucuronide.  for  and c h a r a c t e r i s a t i o n  from the u r i n e have r e v e a l e d the proposed  structure  of  i n pharmacokinetic  half-life  disposition  carbamoyl  A c i d or enzyme h y d r o l y s i s s t u d i e s of u r i n e have shown the  suggesting a s t e r e o s e l e c t i v e glucuronidation  Differences  of  d i r e c t mass  tocainide  presence of a l a r g e excess of a g l u c u r o n i d e d e r i v e d  such as  the drug removed by  a l s o has been d e t e r m i n e d .  tocainide  isomer,  the  studies i n a patient with renal  P r e l i m i n a r y s t u d i e s on the i s o l a t i o n  spectral  this  the d r u g .  dysfunction revealed increased  These  mechanism of e x c r e t i o n of  the s a l i v a which cannot be e x p l a i n e d  T o c a i n i d e enantiomer d i s p o s i t i o n  are  or u r i n e .  of  and c l e a r a n c e  this  drug.  parameters of also point  from the  R(-)  pathway.  the i n d i v i d u a l  enantiomers,  to a s t e r e o s e l e c t i v e  -  206  -  REFERENCES  Adams SS, B r e s l o f f P and Mason CG: P h a r m a c o l o g i c a l D i f f e r e n c e s between the O p t i c a l Isomers of I b u p r o f e n : Evidence f o r M e t a b o l i c I n v e r s i o n of the (-)Isomer. 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