TOCAINIDE: E.C.D.-G.L.C. ANALYSIS PHARMACOKINETICS AND DRUG INTERACTIONS B.Pharm,'University of Madras, 1972 M.Pharm, B i r l a Inst i tu te of Technology and Science, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) Div is ion of Pharmaceutics We accept th is thesis as conforming to the required standard by RAMAN KATARAMANAN in THE UNIVERSITY OF BRITISH COLUMBIA January 1979 Raman Venkataramanan, 1978 In presenting th i s thes is in p a r t i a l f u l f i l m e n t , o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f i nanc ia l gain sha l l not be allowed without my written permission. Department of Pharmaceutics The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 January 12 th,1979. / - i i -ABSTRACT Toca in i de , a s t r u c t u r a l analog of l i d o c a i n e i s an o r a l l y e f f e c t i v e an t i a r rhy thm i c agent. From the s t r u c t u r e i t can be pos tu la ted tha t t o c a i n i d e w i l l undergo metabol ism, p r i m a r i l y by o x i d a t i v e processes . Microsomal en-zyme systems respons i b l e f o r such o x i d a t i v e processes have been shown to be induced or i n h i b i t e d by a number of f a c t o r s . The d i s p o s i t i o n of a number of an t i a r rhy thmic agents has been shown to be i n f l uenced by co-admin i s te red drugs. The major o b j e c t i v e of t h i s study was to i n v e s t i g a t e the suscep-t i b i l i t y of t o c a i n i d e to enzyme induc ing and i n h i b i t i n g agents . An e l e c t r on capture de tec to r gas chromatographic method i n v o l v i n g the format ion of a monoheptaf luorobutyry l d e r i v a t i v e of t o c a i n i d e was developed f o r the a n a l y s i s of t o c a i n i d e i n b i o l o g i c a l f l u i d s . The minimum de tec tab le l e ve l of t o c a i n i d e was 50 peg and the standard curve obta ined was l i n e a r over a range of 100 peg to 3 ng. The e l i m i n a t i o n k i n e t i c s of t o ca i n i de was found to be non - l i nea r over the dosage range s t ud i e d . The area under the plasma concen t ra t i on versus time curve increased i n a d i s -p ropor t i ona te manner w i th an increase in the dose adm in i s t e red . A g rea te r than 100% inc rease in the dose excreted as the i n t a c t t o ca i n i de in u r i ne suggested that the dose dependency of t o c a i n i d e e l i m i n a t i o n i s due to sa tu rab le metabol ism. Fo l l ow ing adm in i s t r a t i o n of t o c a i n i d e (15 mg/kg) to r a t s , 15-20% of the dose was excreted as the i n t a c t drug. An a dd i t i o n a l 20% of the dose was excreted as the a c i d hydro lysab le conjugates of t o c a i n i d e . Th is i s an under e s t ima t i on of the t o t a l amount of the con-jugates present , as l a t e r s tud i e s revea led i n s t a b i l i t y of t o c a i n i d e under the cond i t i on s used f o r a c i d h y d r o l y s i s . Hyd ro l y s i s w i th 3-g lucuron idase showed approx imate ly 40% of the conjugate to be a g lucuron ide con jugate . - i i i -The enzyme hyd r o l y s i s mediated by 3-g lucuronidase was blocked by i t s spe-c i f i c i n h i b i t o r 1:4 saccharo- l ac tone . During gas chromatography-mass spectrometry s tud ies of the u r i n e , a deaminated t o c a i n i d e , dimethyl a n i l i n e (xy l i d ine) and a c y c l i c hydantoin d e r i v a t i v e of t o c a i n i d e were a l s o observed to be present as metabo l i t es in r a t u r i n e . A m u l t i p l e a n a l y t i c a l scheme developed,conf i rmed tha t a c y c l i c hydantoin d e r i v a t i v e o f t o c a i n i d e which . was reported to be a "metabonate" i n man, was present .as a me tabo l i t e . i n r a t s . Pretreatment of the r a t s w i th phenobarb i ta l (70 mg/kg f o r seven days) markedly reduced the h a l f - l i f e of t o c a i n i d e (20 mg/kg) by t w o f o l d . Th is was due to an i nduc t i on of the metabol ism of t o c a i n i d e , as shown by a s i g n i f i c a n t reduc t ion i n the percent of dose excreted as i n t a c t t o ca i n i de in r a t s . The extent of i nduc t i on was more pronounced f o l l ow i ng o ra l ad -m i n i s t r a t i o n of t o ca i n i de as compared to an int ravenous adm in i s t r a t i o n of t o c a i n i d e . The reduc t i on in the percent of dose excreted as the a c i d hydro-l y s ab l e conjugate suggested tha t pathways o ther than con jugat ing systems are probably induced to a g rea te r ex tent than con jugat ing systems. SKF 525-A (50 mg/kg admin is tered 40 minutes p r i o r to t o ca i n i de ) pretreatment markedly prolonged the h a l f - l i f e of t o c a i n i d e (15 mg/kg). The i n h i b i t i o n of t o c a i n i d e metabolism by SKF 525-A was r e f l e c t e d i n terms of the inc rease i n the per-cent o f dose excre ted as i n t a c t t o c a i n i d e . The ex tent of i n h i b i t i o n o f the metabolism was g rea te r f o l l ow i ng a 15 rng/kg dose-as compared to a 20, mg/kg dose. Th is observa t i on supports the ex i s tence of a sa tu rab le metabo l i c pathway f o r the e l i m i n a t i o n of t o ca i n i de i n r a t s . Experiments were c a r r i e d out to determine the e f f e c t of a; compet i t i ve i n h i b i t o r of g lucuron ide con-juga t i ng pathways on the e l i m i n a t i o n of t o c a i n i d e . The observa t i on of an - i v -increased ex c r e t i on of the percent of dose as the i n t a c t t o c a i n i d e suggests compet i t i ve i n h i b i t i o n of t o c a i n i d e metabol ism. However, the extent of i n h i b i t i o n was found to depend upon the route o f a dm in i s t r a t i o n as we l l as the pretreatment schedule used. S ince i n humans, g lucuron ide con jugat ion accounts f o r a g rea te r percent of the dose admin i s t e red , sa l i cy!amide would be expected to have a more pronounced e f f e c t on t o ca i n i de metabol ism i n human. I t can be concluded tha t the d i s p o s i t i o n of t o c a i n i d e i s s u s c ep t i b l e to both enzyme induc ing and enzyme i n h i b i t i n g agents in r a t s . The r e s u l t s obta ined in the present study po in t s to the neces s i t y f o r c a r r y i n g out s i m i l a r experiments in humans so tha t the pa t i en t s cou ld ob ta in maximum bene f i t s of t o c a i n i d e therapy. Fur ther more * caut ion should be exe r c i s ed i n i n t e r p r e t i n g r e s u l t s when nonphys io l og i ca l so l ven ts are used to admin i s te r p o o r l y . s o l u b l e drugs . - v -TABLE OF CONTENTS Chapter Page ABSTRACT i i LIST OF TABLES x i v LIST OF FIGURES xv i LIST OF SCHEMES • x i x SYMBOLS AND ABBREVIATIONS xx INTRODUCTION 1 I . LITERATURE SURVEY 6 A. K i n e t i c s and Metabol ism of Toca in ide and i t s Analogs 6 1. Pharmacokinet ics of Toca in ide 6 2. K i n e t i c s of Pharmacologica l E f f e c t of Toca in ide 11 3. P r o t e i n B ind ing of Toca in ide 12 4. S t r u c t u r e - a c t i v i t y - Pharmacok inet ics 12 5. E l im i n a t i o n of Toca in ide 15 6. T o x i c i t y S tud ies on Toca in ide 20 a) Animal T o x i c i t y S tud ies 20 b) Human T o x i c i t y S tud ies 20 B. Drug Level Measurements i n B i o l o g i c a l F l u i d s 21 C. K i n e t i c s and Metabol ism 22 D. Enzyme Induct ion 24 E. Enzyme I n h i b i t i o n 27 F. E f f e c t of Compet i t ive I n h i b i t i o n of Glucuronide Conjugat ing Enzymes 29 - v i -Chapter Page I I . EXPERIMENTAL 31 A. Ma te r i a l 31 1. Chemicals 31 2. Reagents 31 3. So lvents 32 4. Ma t e r i a l s f o r Animal Surgery 32 B. I d e n t i f i c a t i o n and P u r i t y Determinat ion 33 1. Phys ico-chemica l Methods 33 2. Chromatographic Methods 33 a) G.L.C. 33 b) H.P.L.C. 33 C. P repara t i on of Base and Other Stock So l u t i on s 34 1. P repara t i on o f Toca in ide Base 34 2. P repara t i on of Aqueous So l u t i o n of Toca in ide Hydroch lor ide 34 3. P repara t i on of Toca in ide Base i n Benzene 35 4. P repara t i on of In te rna l Standard So l u t i o n 35 D. P r e l im i na r y F . I .D . -G . L .C . Ana l y s i s 35 1. Column S e l e c t i o n 36 a) 10% UCW 982 36 b) 3% OV-25 37 c) 10% OV-101 37 d) 3% OV-17 38 2. D e r i v a t i z a t i o n 38 3. Op t im i za t i on o f G.L.C. Cond i t i ons 42 4. P repara t i on of the So l u t i o n f o r Standard Curve 42 a) Toca in ide Hydroch lor ide So l u t i o n 42 b) W-49167 Hydroch lo r ide So l u t i o n 43 - v i i -Chapter Page E. P r e l im i na r y E .C.D.-G.L .C. S tud ies 43 1. S e l e c t i o n of D e r i v a t i s i n g Agents 43 2. S e l e c t i o n of an In te rna l Standard 45 3. Op t im i za t i on of De r i v a t i v e Formation 46 . a) K i n e t i c s of De r i v a t i v e Formation 48 b) S e l e c t i o n of So lvent f o r React ion 48 c) S e l e c t i o n o f Volume of the Reagent Used 50 4. Removal of the Excess D e r i v a t i z i n g Agent 50 5. S t a b i l i t y of the De r i v a t i v e Formed 50 6. Conf i rmat ion of the S t r u c t u r e of the De r i v a t i v e Formed 52 7. Op t im i za t i on of G.L.C. Cond i t i ons 52 a) Column S e l e c t i o n 52 b) Op t im i za t i on of Some G.L.C. Parameters 53 8. Op t im i za t i on of E x t r a c t i o n 54 a) S e l e c t i o n of So lvent f o r E x t r a c t i o n 54 b) Phase-volume Rat io 54 c) M u l t i p l e E x t r a c t i o n vs S i ng l e E x t r a c t i o n 54 F. Recovery S tud ies 55 1. Standard Curve of Toca in ide Base 55 2. Standard Curve of the Plasma/Urine E x t r a c t 55 G. Faecal Exc re t i on Stud ies 56 H. S t a b i l i t y o f Toca in ide a t 55° in Hexane 57 I . Opt imized A n a l y t i c a l Cond i t i ons 58 J . Drugs, Doses and Dosage Forms 60 K. Animal Study 61 1. Plasma Level Study 64 - v i i i -Chapter Page a) P repara t i on o f the Cannula 64 b) Su rg i ca l Procedure 65 c) Exper imentat ion 67 2. U r ina ry Exc re t i on S tud ies 68 L. Determinat ion of the Presence of Toca in ide Conjugate 69 1. Ac i d Hyd ro l y s i s 69 2. 3-G1ucuronidase Hyd ro l y s i s 69 3. E f f e c t o f 1:4 Saccharo-Lactone on the Enzymatic Hyd ro l y s i s of Toca in ide Conjugate 69 4. Op t im i za t i on of Enzyme Hyd ro l y s i s 70 a) E f f e c t o f Enzyme (glucurase®) Concen-t r a t i o n on the Hyd ro l y s i s o f the Glucuronide Conjugate 70 b) Comparison o f the E f f e c t of Acetate Bu f f e r vs the Use of D i l u t e Hydroch lo r i c Ac i d to Ad jus t the pH of the Incubat ion Medium 70 c) E f f e c t o f the Source of 3-G1ucuron idase on the Hyd ro l y s i s o f the Conjugate Toca in ide 71 R R i ) G l u su l a se vs g lucurase 71 R i i ) G lucurase vs 3 - g l u cu ron i da se (mol lusk) 71 d) E f f e c t o f Chloroform on the Enzymatic Hyd ro l y s i s o f Toca in ide Conjugate 72 5. Op t im i za t i on o f Ac i d Hyd ro l y s i s 72 a) Incubat ion w i th hyd roch l o r i c a c i d i n an oven a t 100° 72 b) Hyd ro l y s i s by Au toc l av i ng w i th Ac i d 72 6. S t a b i l i t y o f Toca in ide Hydroch lo r ide So l u t i o n a t 100° 73 7. S t a b i l i t y of an Aqueous So l u t i o n of Toca in ide in the Presence of Added Urea 73 M. M u l t i p l e A n a l y t i c a l Scheme f o r Determining the O r i g i n and Nature of the C y c l i c Me tabo l i t e 73 i x -Chapter Page 1. E x t r a c t i o n Procedure 73 2. G.C.-M.S. Cond i t i ons 74 3. D e r i v a t i z a t i o n 76 a) Methy la t i on 76 b) A c y l a t i o n 77 N. Treatment of Data 77 I I I . RESULTS AND DISCUSSION 79 A. Conf i rmat ion of the P u r i t y and the I d e n t i t y of the Ma t e r i a l s 79 1. P u r i t y and I d e n t i t y of the Chemicals 79 2. P u r i t y of the So lvent 79 B. Chromatographic Ana l y s i s of Toca in ide 83 1. F . I .D . -G . L .C . Ana l y s i s 83 2. E . C D . - G . L . C . Ana l y s i s 87 a) L i m i t a t i o n of F . I .D . -G . L .C . Ana l y s i s 87 b) Optimal Cond i t i ons f o r D e r i v a t i z a t i o n 91 i ) S e l e c t i o n of d e r i v a t i z i n g agent 91 i i ) So lvent f o r the r e a c t i o n 92 i i i ) K i n e t i c s of d e r i v a t i v e format ion 92 i v ) Removal o f excess reagent 93 v) S t a b i l i t y o f the d e r i v a t i v e 93 v i ) S t r u c t u r e of the d e r i v a t i v e formed 93 c.) Opt im iza t i on , of G.L.C. Cond i t i ons 94 i ) E f f e c t of d i f f e r e n t s t a t i o na r y phases 94 i i ) E f f e c t of column temperature and : c a r r i e r gas f low TOO i i i ) E f f e c t of i n j e c t i o n por t temperature 100 i v ) E f f e c t of de tec to r temperature 100 d) Op t im i za t i on of E x t r a c t i o n 102 - X -Chapter Page i ) So lvent e f f i c i e n c y 102 i i ) Recovery s tud i e s 102 e) G.L.C. of Plasma and Ur ine Samples 107 f ) Ana l y s i s of Faecal Samples 107 g) V a r i a t i o n i n the A n a l y t i c a l Methodology 109 h) In te r fe rence by Other Compounds 109 i ) Comparison o f E . C D . - G . L . C . vs F . I .D . -G . L .C . Ana l y s i s "109 C. Se l e c t i o n of Animal Model 112 D. Pharmacokinet ics o f Toca in ide 114 1. Plasma Level S tud ies 114 2. U r i na ry Exc re t i on S tud ies 125 a) Species D i f f e r ence i n the U r i na ry Exc r e t i on of Toca in ide 125 b) E f f e c t of Dose and Route of Adm in i s t r a t i o n Ur ina ry Exc re t i on of Toca in ide 127 3. Ana l y s i s of the Po s s i b l e Reason f o r the Non-l i n e a r i t y t r i ' t he K i n e t i c s of Toca in ide 131 a) N o n - l i n e a r i t y due to Absorp t i on 132 b) N o n - l i n e a r i t y due to D i s t r i b u t i o n a l Processes 132 c) N o n - l i n e a r i t y due to E l im i n a t i o n Processes 135 i ) Ex c r e t i on through kidney 135 i i ) B i l i a r y e x c r e t i on 137 i i i ) Metabol ism = Evidence f o r s a t u r a t i o n of metabol ism 139 i v ) -Add i t i ona l evidence f o r the s a t u r a t i o n of me tabo l i z i ng enzyme systems ' 141. E. Metabol ism 142 1. Conjugat ion as a Metabo l i c Pathway 142 2. Determinat ion o f the Presence of Conjugates of Toca in ide 144 3. Op t im i za t i on of the Enzymatic Hyd ro l y s i s 147 - x i -Chapter Page a) E f f e c t of Amount of Enzyme on the Hyd ro l y s i s of Toca in ide Conjugate 147 b) E f f e c t of Acetate Bu f f e r ys Hydroch lo r i c Ac i d on the Enzyme Hyd ro l y s i s 147 c) E f f e c t of pH on the Enzyme Hyd ro l y s i s of Toca in ide Conjugate 150 d) E f f e c t of the Source of Enzyme on the Hyd ro l y s i s of Toca in ide Conjugate 150 e) E f f e c t of Chloroform on the Hyd ro l y s i s Mediated by 3-g lucuron idase 150 f ) E f f e c t of Time of Incubat ion on the Enzyme Hyd ro l y s i s 153 g) Opt imized Cond i t i ons of the Enzyme Hyd ro l y s i s 153 4. Ac i d Hyd ro l y s i s 153 a) A c i d Hyd ro l y s i s of the Conjugate a t 100° 153 b) Ac id Hyd ro l y s i s of the Conjugate by Auto-c l a v i n g 155 5. S t a b i l i t y of an Aqueous So l u t i o n of Toca in ide Hydroch lor ide 155 6. S t a b i l i t y of an Aqueous So l u t i o n of Toca in ide Hydroch lor ide in Presence of Added Urea 155 7. Presence of Conjugate other than G lucuron ide Conjugate 160 8. Add i t i o na l Me tabo l i t e s of Toca in ide 160 9. I d e n t i t y and O r i g i n of the C y c l i c Compound 163 a) C y c l i c Me tabo l i t e s of Toca in ide Analog 163 b) Development of an A n a l y t i c a l Scheme 168 c) G.L.C.-Mass Spectrometry 170 d) I d e n t i f i c a t i o n of the S t ru c tu re of the C y c l i c Compound 170 e) Me tabo l i t e or Metabonate 177 f ) Po s s i b l e O r i g i n of the C y c l i c Compound 181 F. Enzyme Induct ion 184 1. Induct ion of Drug Me tabo l i z i ng Enzyme by Phenobarb i ta l 184 - x i i -Chapter Page 2. Se l e c t i o n of Dose and Dosing Schedule of Phenobarbit 'al 189 3. S e l e c t i o n o f Dose of Toca in ide 189 4. E f f e c t of Rhenobarb i ta l on the D i s p o s i t i o n of Toca in ide 190 a) Plasma Level S tud ies 190 b) U r ina ry Exc re t i on S tud ies 190 c) E f f e c t o f Phenobarb i ta l on the Metabo l i t e Formation 194 G. Enzyme I n h i b i t i o n 198 1. E f f e c t of SKF 525-A on Drug Me tabo l i z i ng Enzymes 198 2. Pretreatment Schedule 201 3. S e l e c t i o n of Toca in ide Dose 202 4. E f f e c t of SKF 525-A on the D i s p o s i t i o n of Toca in ide 202 a) Plasma Level S tud ies 202 b) Ur inary Exc re t i on Stud ies 205 c) E f f e c t of Route of Adm in i s t r a t i o n of Toca in ide on the I n h i b i t i o n of Metabol ism 208 d) E f f e c t of SKF 525-A on the E l im i n a t i o n of Toca in ide by Rats P re t rea ted w i th Pheno-barb i t a l 208, H. E f f e c t of Compet i t ive I n h i b i t i o n of Metabo l i c Pathway on the D i s p o s i t i o n of Toca in ide 210 1. E f f e c t of Sa l i c y l am ide on Drug D i s p o s i t i o n 210 2. P repa ra t i on of Dosage Form o f Sa l i c y l am ide 210 3. Route of Adm in i s t r a t i o n of Sa l i c y l am ide and Toca in ide 210 4. E f f e c t of Sa l i c y l am ide on the D i s p o s i t i o n of Toca in ide 211 a) U r i na ry Exc r e t i on S tud ies 211 - x i i i -Chapter Page SUMMARY AND CONCLUSIONS 214 REFERENCES 217 APPENDIX - A 232 APPENDIX - B 236 APPENDIX - C 239 - x i v -LIST OF TABLES Table Page I Pharmacokinet ic parameters o f - t o c a i n i d e i n normal sub-j e c t s f o l l ow i ng in t ravenous adm in i s t r a t i o n 8 II Summary of t o c a i n i d e pharmacokinet ic data a t steady s ta te 10 I I I a . S t r u c t u r a l analogs of l i d o c a i n e 13 b. S t r u c t u r a l analogs o f t o ca i n i de 14 IV Comparison of the k i n e t i c p r ope r t i e s of t o ca i n i de and l i d o c a i n e in man 16 V Pharmacokinet ic data f o r d i f f e r e n t am inoacy l xy l i d i nes f o l l ow i n g in t ravenous i n f u s i o n i n t o dogs 17 VI K i n e t i c s of t o c a i n i d e i n Beagle dogs 18 VII Treatment and pretreatment schedule 62 VII I Es t imat i on of t o ca i n i de by flame i o n i z a t i o n de tec to r gas chromatography (standard curve) 90 IX Response of t o ca i n i de d e r i v a t i v e and the i n t e r n a l standard i n d i f f e r e n t s t a t i o n a r y phases 98 X E f f e c t o f i n j e c t i o n port temperature on the response of t o c a i n i d e d e r i v a t i v e 101 XI E f f e c t o f de tec to r temperature on the response of t o ca i n i de d e r i v a t i v e 103 XII Es t imat ion of t o ca i n i de by e l e c t r on capture de tec to r gas chromatography (standard curve) 103 XII I Recovery o f t o c a i n i d e from r a t plasma as determined by e l e c t r on capture de tec to r gas chromatography 106 XIV Comparison of the flame i o n i z a t i o n de tec to r gas chromato-graphy and the e l e c t r on capture detector , gas chromatography 111 XV Plasma concent ra t i on .vs time f o l l ow i n g adm in i s t r a t i o n of 10 mg/kg t o ca i n i d e to 4 r a t s 116 XVI Plasma c o n c e n t r a t i o n ^ time f o l l ow i ng adm in i s t r a t i o n of 20 mg/kg t o ca i n i de to 4 r a t s 119 XVII Pharmacokinet ic parameters of t o c a i n i d e in r a t f o l l ow i ng in t ravenous adm in i s t r a t i o n of 5, 10, 15, 20 and 40 mg/kg t o ca i n i d e 121 - X V -Table Page XVIII Add i t i o na l pharmacokinet ic parameters of t o ca i n i de in r a t f o l l ow i ng adm in i s t r a t i o n of 5, 10, 15, 20 and 40 mg/kg t o ca i n i de 124 XIX U r i na ry e x c r e t i on of t o c a i n i d e i n var ious spec ies of animals f o l l ow i ng o ra l a dm in i s t r a t i o n 126 XX E f f e c t of d i f f e r e n t doses of t o c a i n i d e on the u r i n a r y pH 138 XXI " E f f e c t of d i f f e r e n t sources of enzymes on the h yd r o l y s i s of t o ca i n i de conjugate - a . g lucurase vs g lusu l ase 152 b. - g lusurase vs 3-glucuron.ide. (mol lusk) XXV E f f e c t o f du ra t i on of i n cuba t i on and s t reng th o f a c i d on the h yd r o l y s i s of t o c a i n i d e conjugate 156 XXVI E f f e c t of phenobarb i ta l pretreatment on the k i n e t i c s of t o c a i n i d e 192 XXVII E f f e c t of phenobarb i ta l pretreatment on the u r i ne pH va lues i n r a t s 195 XXVIII E f f e c t of phenobarb i ta l pretreatment on the amount o f t o c a i n i d e conjugate excre ted i n u r ine 196 XXIX E f f e c t of SFK 525-A pretreatment on the k i n e t i c s of t o c a i n i d e 204 XXX E f f e c t of SKF 525-A pretreatment on the u r i ne pH va lues i n r a t 209 XXXI E f f e c t o f sa l icy1 amide pretreatment on the u r i n a r y ex c r e t i on of i n t a c t t o c a i n i d e 212 - x v i . -LIST OF FIGURES F igure Page 1 S t r u c t u r e o f l i d o c a i n e and t o c a i n i d e 7 2 Gas chromatogram showing the response of t o ca i n i de (a) and i n t e r n a l standard (b) i n d i f f e r e n t columns A - 3% OV-25; B - 5% OV-101; C-5% OV-101; D - 3% OV-17 39 3 Gas chromatograms o f im idazo le [A] and d e r i v a t i z e d t o ca i n i d e (a) and the i n t e r n a l standard (b) [B] in 3% OV-17 column 41 4 Gas chromatograms of d i f f e r e n t i n t e r n a l standards eva luated W-49167 [A],MEGX [B] and Diazepam [C] 47 5 K i n e t i c s of d e r i v a t i v e format ion 49 6 S t a b i l i t y of the d e r i v a t i v e formed a t room temperature 51 7 Cannula used f o r j u gu l a r ve in cannu la t i on 66 8 D i f f e r e n t i a l scanning c a l o r ime t r y o f t o c a i n i d e hydro-c h l o r i d e - 80 9 D i f f e r e n t i a l scanning c a l o r ime t r y of t o c a i n i d e base. A. Ex t rac ted using methylene c h l o r i d e B. Ex t rac ted us ing benzene 81 10 High pressure l i q u i d chromatogram of t o ca i n i de 82 11 Flame i o n i z a t i o n de tec to r -gas chromatogram of t o ca i n i de (a) and W-49167 (b) from As t r a Pharmaceut ica l P roduc ts , Inc . 86 12 Flame i o n i z a t i o n detec tor -gas chromatogram; o f t o c a i n i d e (a) and W-49167 (b) mod i f i ed method 88 13 Plasma l e v e l s of t o c a i n i d e i n a p a t i e n t r e c e i v i n g o ra l t o c a i n i d e hydroch lo r i de . 89 14 Mass spectrum o f t o ca i n i de d e r i v a t i v e obta ined by G .L .C . -mass spectrometry a f t e r e x t r a c t i o n o f plasma from r a t s dosed w i th t o ca i n i de (20 mg/kg) 95 15 E l e c t r on capture de tec tor -gas chromatogram of a-Bromonaphthalene (a) and t o ca i n i d e d e r i v a t i v e (b) i n d i f f e r e n t columns A - 3% OV-17 B - 10% 0V-7 C - 5% 0V-101 D - 5% SP2250 99 16 E x t r a c t i o n e f f i c i e n c y of d i f f e r e n t so l ven ts 104 - x v i i -F igure Page 17 E l e c t r on capture de tec to r -gas chromatogram of a-Bromonaphthalene (a) and t o ca i n i d e d e r i v a t i v e (b) i n 3% OV-17. A - Blank plasma B - plasma e x t r a c t C - Blank u r ine D - Ur ine e x t r a c t E - Blank faeces F - Aqueous e x t r a c t 108 18 E l e c t r on capture de tec to r -gas chromatogram of some drugs to t e s t f o r i n t e r f e r en ce in a n a l y s i s . A -G r i s e o f u l v i n (oven temperature 280°); B - Meto-c lopramide; C - S a l i c y l am i de ; D - SKF 525-A; E - Diazepam; and F - G l y c i ne x y l i l i d e 110 19 Semi logar i thmic p l o t of mean ( ± S.D.) plasma concen-t r a t i o n vs time f o l l ow i ng in t ravenous a dm i n i s t r a t i o n of t o ca i n i de (10 mg/kg) to 4 r a t s 115 20 Semi logar i thmic p l o t of mean ( ± S.D.) plasma concen-t r a t i o n vs time f o l l ow i n g in t ravenous a dm i n i s t r a t i o n of t o c a i n i d e (20 mg/kg) to 4 r a t s 118 21 Semi logar i thmic p l o t o f plasma concen t ra t i on vs t ime f o l l ow i ng in t ravenous adm in i s t r a t i o n of 5, 10, 15 and 20 mg/Kg of t o c a i n i d e . • 120 22 P l o t of area under the plasma concen t ra t i on y_s time 122 oo curve ( f Cpdt = AUC) aga ins t dose o f t o c a i n i d e admin i s te red i n t r avenous l y to r a t s 23 E f f e c t of route o f a dm in i s t r a t i o n on the u r i n a r y e x c r e t i on o f t o ca i n i de f o l l o w i n g d i f f e r e n t doses 128 24 E f f e c t of d i f f e r e n t doses of t o c a i n i d e admin i s te red i n t r a -venous ly , on the u r i n a r y ex c r e t i on of i n t a c t t o c a i n i d e 129 in r a t s 25 B i o a v a i l a b i l i t y of t o c a i n i d e , f o l l ow i ng o r a l and i n t r a -pe r i t onea l a d m i n i s t r a t i o n , c a l c u l a t e d us ing u r i n a r y ex c r e t i on data 130 26 Theo re t i c a l p l o t of the e f f e c t o f non - l i n ea r plasma p ro t e i n b ind ing on the plasma l e v e l s o f drugs 133 A - Case 1 B - Case 2 27 E f f e c t of d i f f e r e n t amounts of enzyme on the h yd r o l y s i s o f t o c a i n i d e conjugate 148 28 Ac i d h yd r o l y s i s of t o c a i n i d e conjugate i n oven a t 100° 154 - xv i i i -F igure Page 29 Ac i d h yd r o l y s i s o f t o ca i n i de conjugate in an autoc lave 157 30 S t a b i l i t y of aqueous s o l u t i o n o f t o ca i n i de a t 100° f o r 1 hour i n presence of d i f f e r e n t s t rengths of a c i d 158 31 S t a b i l i t y o f aqueous s o l u t i o n of t o ca i n i de a t 100° f o r d i f f e r e n t per iods of t ime 159 32 Fragmentat ion pat te rn and pos tu la ted s t r u c t u r e of a metabo l i t e o f t o c a i n i d e (A) 162 33 Fragmentation pa t te rn and pos tu la ted s t r u c t u r e of a metabo l i t e of t o c a i n i d e (B) 164 34 Fragmentation pa t te rn and pos tu l a ted s t r u c t u r e of a metabo l i t e o f t o c a i n i d e (C) 165 35 S t ru c tu re of the c y c l i c toca-ini'de d e r i v a t i v e : (A) and ' o ther c y c l i c me tabo l i t e s of t o c a i n i d e ana logs . 167 36 Gas-chromatogram ; of methylene c h l o r i d e e x t r a c t of u r ine sample i n SILAR-10C column * 171 37 Gas-chromatogram :of methanol ic e x t r a c t of f reeze d r i ed ur ine sample i n SILAR-10C column 172 38 Mass f ragmentat ion pa t te rn of the c y c l i c metabo l i t e (F) of t o ca i n i de 175 39 E f f e c t of phenobarb i ta l pretreatment on the plasma con-cen t r a t i on vs^ t ime data f o l l ow i n g adm in i s t r a t i o n o f t o ca i n i de (20 mg/kg) 191 40 E f f e c t of phenobarb i ta l on the u r i na r y ex c r e t i on of i n t a c t t o c a i n i d e f o l l ow i ng 20 mg/kg of t o c a i n i d e admin i s te red o r a l l y and i n t r avenous l y 193 41 E f f e c t of SKF 525-A pretreatment on the plasma concen-t r a t i o n vs time f o l l ow i n g in t ravenous adm in i s t r a t i o n of t o ca i n i de (15 mg/kg) 203 42 E f f e c t of SKF 525-A pretreatment on the u r i n a r y e x c r e t i on of i n t a c t t o c a i n i d e , f o l l o w i n g adm in i s t r a t i o n of t o ca i n i de (15 mg/kg) g iven o r a l l y and i n t r avenous l y 206 43 E f f e c t o f SKF 525-A pretreatment on the u r i na r y e x c r e t i o n of i n t a c t t o ca i n i de f o l l ow i n g o ra l a dm i n i s t r a t i o n of 20 mg/kg of t o c a i n i d e 207 - x i x -LIST OF SCHEMES Scheme Page 1 E l e c t r on capture de tec to r gas chromatographic a n a l y s i s of t o c a i n i d e - Procedure 59 2 M u l t i p l e a n a l y t i c a l scheme f o r determin ing the o r i g i n and nature of the c y c l i c metabo l i t e 74 3 Flame i o n i z a t i o n de tec to r gas chromatographic a n a l y s i s of t o c a i n i d e - procedure - developed by As t r a Pharmaceut ica l Products Inc . 84 4 Mod i f i ed f lame i o n i z a t i o n de tec to r gas chromatographic a n a l y s i s of t o ca i n i de 85 5 D e r i v a t i z a t i o n of t o c a i n i d e w i t h hep ta f1uorobuty r i c anhydr ide 96 6 Mass f ragmentat ion pa t t e rn of t o c a i n i d e d e r i v a t i v e 97 7 M u l t i p l e a n a l y t i c a l scheme f o r determin ing the o r i g i n and nature of the c y c l i c metabo l i t e 169 8 Mass f ragmentat ion pa t te rn o f t o c a i n i d e 174 9 Mass f ragmentat ion pa t t e rn o f c y c l i c compound (A) 176 10 Summary data on the o r i g i n and nature of c y c l i c com-pound (A) i n r a t s 179 - XX -SYMBOLS AND ABBREVIATIONS a ^d i s t r i bu t i on ra te constant AUC area under the plasma l e v e l vs time curve (3 hybr id e l i m i n a t i o n ra te constant ( d i s p o s i t i o n r a t e constant C . I . chemical i o n i z a t i o n C 1 B t o t a l body c learance C 1 N R non-renal c learance C 1 R renal c learance D . S . C . d i f f e r e n t i a l scanning c a l o r ime te r E . C . D . e l e c t r on capture de tec to r E : I . e l e c t r on impact F . I . D . f lame i o n i z a t i o n de te c t o r G . C . - M . S . gas chromatograph-mass spectrometer • G . L . C . gas l i q u i d chromatograph HFBA hep t a f l u o r obu t y r i c anhydr ide HFBI hep t a f l u o r obu t y r i c im idazo le HPLC high performance l i q u i d chromatograph i . d . i n t e r n a l d iameter i .p. i n t r a p e r i t o n e a l i . v . in t ravenous K 1 2 t r an spo r t r a te constant from compartment 1 to compartment 2 K 2 1 t r anspo r t ra te constant from compartment 2 to compartment 1 K E e l i m i n a t i o n ra te constant from compartment 1 MEGX monoe thy l gy l c i nexy l i d i de - xx i -Methelute t r ime t hy l a n i l i n um hydroxide Methyl - 8 dimethy l formide dimethyl a ce ta l NADPH c y t . c reductase n i co t inamide adenine diphosphate cytochrome :c reductase PCT plasma, concen t ra t i on versus time curve PFBC pentaf luorobenzoy l c h l o r i d e TMS-TFA N-methyl N - t r i m e t h y l s i l y l t r i f l u o r o a c e t am i de UDP-GAD u r i d i n e diphosphate g lucu ron i c a c i d dehydrogenase UDP-GT u r i d i n e diphosphate g lucurony l t r ans f e r a se V c volume of the cen t r a l compartment volume of d i s t r i b u t i o n V d s s volume o f d i s t r i b u t i o n a t steady s t a t e V T volume o f the t i s s u e compartment - x x i i -ACKNOWLEDGEMENT The author wishes to acknowledge Dr. Axel son f o r suggest ing the problem and f o r h i s help dur ing the course of s tudy. The author i s g r a t e f u l to Drs. Abbot t , M i t c h e l l , Godo lph in , M c N e i l l , Hassman and B i l l Campbell J r . f o r t h e i r guidance and suggest ion dur ing the course of t h i s work. A word of thanks go to J ama l i , Caro l L a i , Tarn, Connie, Roland and" Kang. f o r help and a s s i s t a n c e . Spec ia l thanks to Helen f o r her constant support throughout the work and i n p a r t i c u l a r her help dur ing animal work. The f i n a n c i a l support prov ided by the U n i v e r s i t y of B r i t i s h Columbia, B.C. Heart Foundation and Canadian Heart Foundation dur ing the course of study i s g r a t e f u l l y acknowledged. The author i s g r a t e f u l to Dr. La lka f o r p rov i d i ng some unpubl ished r e s u l t s and to As t r a Pharmaceut ical P roducts , Inc. f o r p rov i d i ng the drug samples. - x x i i i -TO MUM AND DAD INTRODUCTION Card iac a r rhy thmias , i n p a r t i c u l a r v e n t r i c u l a r a r rhy thmias , o f ten c o n s t i t u t e l i f e th rea ten ing s i t u a t i o n s and t h e i r management i s a g reat cha l l enge to the p h y s i c i a n . Of the commonly used an t i a r rhy thm i c compounds, procainamide was cons idered supe r i o r to qu i n i d i n e i n the treatment o f ven-t r i c u l a r arrhythmias [ 1 ] ; Before the r e c ogn i t i o n of the an t i a r rhy thm i c p rope r t i e s o f 1 idoca ine , procainamide was the drug of cho ice in the t r e a t -ment o f v e n t r i c u l a r e c t op i c beats [ 1 ] . Un fo r tuna te l y , g a s t r i c and CNS d i s tu rbances and a l u p u s - l i k e syndrome were a s soc i a t ed wi th the use of procainamide [ 2 ] . Southworth [3] repor ted the f i r s t case of the success fu l treatment of c a rd i a c arrhythmia w i th 2 ml of 1% 1 idoca ine hydroch lo r i de i n an ep inephr ine hydroch lo r i de (1:100,000) s o l u t i o n i n j e c t ed d i r e c t l y i n to the l e f t v e n t r i c l e of the pa t i en t r e c e i v i n g s imultaneous e l e c t r i c shock. L idoca ine became the most commonly used drug i n i n t e n s i v e and coronary care u n i t s , when i t was shown to be safe and e f f e c t i v e i n the treatment of v e n t r i c u l a r arrhythmias f o l l ow i n g acute myocardia l i n f a r c t i o n . Th is drug i s a l so we l l known f o r i t s a b i l i t y to suppress the premature ven-t r i c u l a r beats which occur in a l a r ge number of pa t i en t s i n coronary care un i t s [ 4 , 5 , 6 ] . L i doca ine has the advantage o f prompt onset o f a c t i o n and minimal haemodynamic d i s tu rbances [ 6 ] . However, there are c e r t a i n drawbacks asso-c i a t e d w i th l i d o c a i n e therapy. Due to i t s very shor t h a l f - l i f e (90 min.) [7] cont inuous in t ravenous i n f u s i o n or repeated systemic i n j e c t i o n i s necessary to ma inta in the des i r ed response. Moreover, the a v a i l a b i l i t y of l i d o c a i n e i s d r a s t i c a l l y reduced when i t i s admin i s te red o r a l l y , due to f i r s t pass metabol ism [ 8 , 9 ] . In a d d i t i o n , c e r t a i n t o x i c e f f e c t s o f - 2 -l i d o c a i n e therapy have been assoc i a ted w i th the metabo l i t es formed [10 ,11 , 12] . The metabo l i t es of l i d o c a i n e , monoe thy l g l y c i nexy l i d i de and g l y c i n e x y l i d i d e have longer h a l f - l i v e s (120 minutes and 10 hours r e s p e c t i v e l y ) compared to tha t of l i d o c a i n e (about 90 minutes) [ 13 ,14 ] . G l y c i ne x y l i d i d e which i s c ha ra c t e r i z ed by a long h a l f - l i f e , accumulates i n s u f f i c i e n t amounts i n the body to produce pharmacologica l as we l l as t o x i c e f f e c t s . Due to the fo rego ing drawbacks w i th l i d o c a i n e , e f f o r t s were d i r e c t ed towards the syn thes i s of an i dea l an t i a r rhy thm i c agent. Th is compound was to be cha r a c t e r i z ed by a prolonged h a l f - l i f e , complete b i o a v a i l -a b i l i t y and absence of f i r s t pass metabol ism. The syn thes i s of t o c a i n i d e by As t r a Pharmaceut ica l P roducts , I n c . , i s the r e s u l t o f a long-term p ro j e c t to develop a 1 i doca i ne -1 i ke drug w i th a prolonged h a l f - l i f e and o ra l e f f e c t i v e n e s s . Toca in ide was e f f e c t i v e in suppress ing a v a r i e t y of exper imental ar rhythmias i n l abo ra t o r y animals [15 ,16 ] . I t i s e f f e c t i v e i n suppress ing the premature v e n t r i c u l a r con-t r a c t i o n (PVCs) i n pa t i en t s [17 ] . I t possesses favourab le pharmacokinet ic p r ope r t i e s such as a long ha l f l i f e [- 11 h r s ] and complete b i o a v a i l a b i l i t y a f t e r o ra l a dm i n i s t r a t i o n [18 ] . P r e sen t l y t h i s compound i s undergoing c l i n i c a l t r i a l s i n the Uni ted S ta tes and such t r i a l s are imminent i n Canada. There i s l i t t l e doubt tha t l i d o c a i n e f a l l s shor t o f be ing an idea l an t i a r rhy thm i c agent due to o ra l i n e f f e c t i v e n e s s , shor t h a l f - l i f e and t o x i c me tabo l i t e s . Toca in ide promises to be a very good s u b s t i t u t e . However, before i t can be accepted as an a l t e r na t i v e ,we need add i t i o n a l i n f o rmat i on about severa l f a c t o r s t ha t may p o t e n t i a l l y a l t e r i t s pharmacokinet ic be-hav io r and hence defeat the purpose f o r which t h i s compound was s yn thes i z ed . The major o b j e c t i v e of t h i s study was to i n v e s t i g a t e the s u s c e p t i -b i l i t y of t o c a i n i d e d i s p o s i t i o n to enzyme induc ing and i n h i b i t i n g agents . - 3 -Card iac pa t i en t s are admin is tered a host o f drugs to t r e a t other a s soc i a t ed problems. The d i s p o s i t i o n of a number of drugs has been shown to be a l t e r e d by the c o - adm in i s t r a t i o n of other drugs [ 19 ] . Depending upon the nature and the extent o f these i n t e r a c t i o n s , t o x i c or subtherapeut i c l e v e l s of the drug and/or metabo l i t e may r e s u l t . Continued suppress ion of PVCs requ i res main-tenance of c e r t a i n minimal plasma l e v e l s of t o c a i n i d e . I t i s , t he re f o re , e s s en t i a l to a s c e r t a i n the extent to which the d i s p o s i t i o n o f t o c a i n i d e can be a l t e r e d by other drugs. The s p e c i f i c aims of the p r o j e c t were as f o l l ow s : 1) A s e n s i t i v e a n a l y t i c a l technique capable of measuring the t rue l e v e l s of i n t a c t drug i n b i o l o g i c a l f l u i d was requ i red f o r c a r r y i n g out pharmacokinet ic s t u d i e s . A f lame i o n i z a t i o n de tec to r gas chromatographic method ( F . I .D . -G . L . C . ) was a v a i l a b l e f o r t h i s purpose.^ Since the use of the r a t as an exper imenta l model n e c e s s a r i l y imposed a r e s t r i c t i o n i n the volume of blood sample tha t cou ld be obta ined dur ing the experiment the F.I.D. assay method cou ld not be used in the present i n v e s t i g a t i o n . There fore , a more s e n s i t i v e , e l e c t r on capture de tec to r gas chromatographic method ( E . C D . - G . L . C . ) which cou ld de tec t picogram q u a n t i t i e s of the drug had to be developed. 2) Upon complet ion of the a n a l y t i c a l procedure development the bas i c pharmacokinet ic p r ope r t i e s of the drug have requ i red s tudy. A knowledge of these parameters wa-s a p r e r e q u i s i t e f o r an eva l ua t i on of the i n f l uence of d i f f e r e n t f a c t o r s such as enzyme i n h i b i t i o n and i n d u c t i o n , on the pharmacokinet ic aspects of t h i s drug. 3) From the s t r u c t u r e i t can be pos tu l a ted tha t t o c a i n i d e w i l l undergo metabol ism p r i m a r i l y by o x i d a t i v e processes . Microsomal enzyme ^ From As t r a :Pharmaceutical Products I n c . , Framingham, Massachuset ts . - 4 -systems r e spons i b l e f o r such o x i d a t i v e processes have been shown to be i nduced / i nh i b i t ed by a number of f a c t o r s [ 20 ] . In a d d i t i o n , a number of drugs have been shown to be po t en t i a l inducers or i n h i b i t o r s of drug meta-b o l i z i n g enzymes [21] , I t i s recognized tha t the metabolism of one drug and hence i t s pharmacologica l a c t i v i t y , can be a l t e r e d by co-admin i s te red drugs. I n i t i a l observa t ions i n humans have i d e n t i f i e d the presence o f N-glucuronide conjugate of t o c a i n i d e i n human u r i ne [ 22 ] . Phenobarb i ta l has been shown to induce g lucuron ide con jugat ing pathways [23-25] . The metabolism of l i d o c a i n e , a s t r u c t u r a l analog o f t o ca i n i de , has been shown to be induced by pretreatment w i th phenobarb i ta l [ 26 ] . In l i g h t of the above i n f o rma t i on , i t was decided to study the e f f e c t o f phenobarb i ta l treatment on the d i s p o s i t i o n of t o c a i n i d e . Phenobarb i ta l a l s o serves as a model to study the e f f e c t of enzyme i nduc t i on on the metabol ism of t o c a i n i d e . 4) I n h i b i t i o n o f drug me tabo l i z i ng enzymes may lead to accumulat ion o f the parent compound and hence po s s i b l e t o x i c s i de e f f e c t s . Astrom [27] has repor ted the r e t a r d i ng e f f e c t of SKF525-A on the r a t e of d isappearance of l i d o c a i n e i n mice. But the metabol ism of p r i l o c a i n e , another s t r u c t u r a l ana log , i s v i r t u a l l y unaf fec ted by SKF 525-A [ 27 ] . I t was, t h e r e f o r e , o f i n t e r e s t to f i n d out the po s s i b l e e f f e c t s of enzyme i n h i b i t i o n on the d i s -p o s i t i o n of t o c a i n i d e . 5) The c apac i t y o f c e r t a i n enzyme systems i nvo l ved i n the b i o -t rans fo rmat ion o f drugs i s l i m i t e d . Co -adm in i s t r a t i on of drugs undergoing metabol ism through capac i t y l i m i t e d metabo l i c pathways w i l l r e s u l t i n com-p e t i t i v e i n h i b i t i o n o f the metabo l i c t r ans f o rma t i on . G lucuron ide conjuga-t i o n has been i d e n t i f i e d as one of the major metabo l i c pathways f o r the - 5 -e l i m i n a t i o n of s a l i c y l am i de [28 ] . The l i m i t e d capac i t y of the enzymes invo lved in t h i s pathway i s ev ident when s a l i c y l am i de a lone i s admin i s te red in h igh doses [ 29 ] . Sa l i c y l am ide has been shown to c ompe t i t i v e l y i n h i b i t the metabol ism o f s a l i c y l i c a c i d and acetaminophen [30 ,31] . S ince g lucuron ide con juga t i on has been i d e n t i f i e d as a b i o t r ans fo rmat i on route in the e l i m i n a t i o n of t o c a i n i d e , i t was decided to study the extent to which s a l i c y l am i de cou ld compete f o r t h i s route o f e l i m i n a t i o n . 6) Toca in ide has been found to undergo a novel route of b i o t r a n s -format ion [22 ] . Ac i d and g lucuron idase h yd r o l y s i s of the ur ine samples y i e l d e d i n t a c t t o c a i n i d e , i n d i c a t i n g the presence of a conjugate . Th is conjugate has been i d e n t i f i e d as a g lucurony l carbamate from the pr imary amine. A l k a l i n e treatment o f t h i s conjugate y i e l d e d a c y c l i c metabonate^ 3 - ( 2 , 6 - x y l y l ) S^-methyl hydantoin [ 22 ] . A s t r u c t u r a l analog of t o c a i n i d e , e t i doca i ne y i e l d s a s i m i l a r hydantoin metabo l i t e i n man.[32]. The presence of t h i s metabo l i t e has been imp l i c a t ed in the lower t o x i c i t y o f e t i do ca i ne compared w i th bup ivaca ine , which does not produce such a metabo l i t e [ 32 ] . Experiments were c a r r i e d out to determine the presence o f conjugates of t o c a i n i d e i n the r a t as we l l as to determine the presence of the c y c l i c hydantoin me tabo l i t e . Experiments were designed to d i s t i n g u i s h whether the hydantoin metabo l i t e was i n f a c t a "metabo l i t e " or a "metabonate". Informat ion obta ined from the present study w i l l po in t out the po t en t i a l areas o f research to be c a r r i e d out i n humans. Metabonate - Metabonate i s a substance which appears to be a metabo l i t e but which i n r e a l i t y i s formed dur ing i s o l a t i o n and/or s to rage . - 6 -I . LITERATURE SURVEY A. KINETICS AND METABOLISM OF TOCAINIDE AND ITS ANALOGS 1. Pharmacokinet ics of Toca in ide Toca in ide (2-amino-2' , 6 ' - p r o p i o n o x y l i d i d e ) ( F i g . 1) i s a pr imary amine analog of l i d o c a i n e . Th is compound possesses a potent an t i a r rhy thm i c e f -f e c t on arrhythmias induced by gradual c o n s t r i c t i o n o f the coronary a r t e r i e s i n dogs [ 16 ] . I t has been shown to be a safe and e f f e c t i v e an t i a r rhy thm i c agent i n pa t i en t s w i th premature v e n t r i c u l a r c on t r a c t i on s [17, 33-36] . De ta i l ed pharmacokinet ic study of t o c a i n i d e in normal human vo lun-tee rs revea led s p e c i f i c pharmacokinet ic advantages of t h i s compound over the p r e sen t l y a v a i l a b l e o ra l an t i a r rhy thm i c agents [ 18 ,37 ] . Table I sum-marizes the k i n e t i c p rope r t i e s of t o c a i n i d e in normal human vo l un t ee r s . Toca in ide was found to be we l l absorbed from the g a s t r o - i n t e s t i n a l t r a c t ( b i o a v a i l a b i l i t y approx imate ly 100%). As the r a t e o f abso rp t i on was very r a p i d , f o l l ow i n g o ra l a d m i n i s t r a t i o n , i t was not po s s i b l e to ass i gn an "order" to the absorp t i on process . The drug e xh i b i t e d l i n e a r pharmacokinet ic p r ope r t i e s over the dose range,10-1000 mg [0.14 mg/kg-14.3 mg/kg] s tud ied [18 ] . The blood l e ve l p r o f i l e , a f t e r in t ravenous adm in i s t r a t i o n was ade-quate ly desc r ibed by a two compartment open model w i th an e l i m i n a t i o n h a l f l i f e o f about 11 hours. The drug was e l im ina ted by both rena l and non-renal pathways. Approx imate ly 40% of the dose g iven o r a l l y was excreted i n the u r ine i n 48 hours. The ra te of abso rp t i on o f t o ca i n i de was de-creased by c o - adm in i s t r a t i o n of food. However, the ex tent of ab so rp t i on , as determined by the area under the blood c o n c e n t r a t i o n ^ time curve - 7 -TOCAINIDE Figure 1: Structure of L idocaine and Tocainide - 8 -Table I Pharmacokinet ic Parameters o f Toca in ide i n Normal Subjec ts Fo l l ow ing Intravenous A d m i n i s t r a t i o n 9 K i n e t i c Parameter Mean Value D i s t r i b u t i o n Rate Constant ( a ) 0.064 m i n u t e - 1 -1 D i s p o s i t i o n Rate Constant (3) 0.0010 minute T rans fe r Rate Constant from Compartment 1 to Compartment 2 ( k 1 2 ) 0.027 minute' •1 T rans fe r Rate Constant from Compartment _1 2 to Compartment 1 ( k ^ ) 0.037 minute" E l im i n a t i o n Rate Constant (K £ ) 0.0018 m i n u t e - 1 Volume o f D i s t r i b u t i o n of Cent ra l Compartment (V c ) 74.7 1. Volume o f D i s t r i b u t i o n a t Steady S ta te (Vd.) 5 S,. 118.5 1. Volume of D i s t r i b u t i o n a t E q u i l i b r i u m (Vd ) a 133.4 1. p Tota l Body Clearance (Clg) 166 ml/minute Renal C learance (C1 R ) 64 ml/minute Non-renal C learance (C-1 N R) 102 ml/minute Model K 1 2 N VT 1 a t room temperature. The res idue was r e - c o n s t i t u t e d w i th 1 ml o f benzene and 5 y l of the benzene s o l u t i o n was i n j e c t e d i n to the gas chromato-graph. The f o l l o w i n g cond i t i on s were used: I n j e c t i o n por t 300°C, oven 180°C, de tec to r 350°C, c a r r i e r gas (95% Argon/5% Methene) 40 ml/min. From the p r e l im i na r y s tud i e s w i th the F . I .D . -G . L . C . , a 3% OV-17 column was se l e c t ed f o r t e s t i n g the d e r i v a t i v e f o rmat i on . With PFBC, i n the absence of a c a t a l y s t , the y i e l d o f the d e r i v a t i v e was very poor. Add i t i o n of a c a t a l y s t , p y r i d i n e , wh i l e i n c r ea s i ng the y i e l d of the d e r i v a t i v e in t roduced a number of background peaks i n the chromatogram. - 45 -Severe d i s tu rbances i n the chromatogram us ing E . C D . - G . L . C . have been a l s o observed by other workers [90] when py r i d i n e was used as a c a t a l y s t . With H F B I , p r e c i p i t a t i o n o f the r e a c t i o n product , im idazo l e , occurred when hexane was used as a so l ven t f o r the r e a c t i o n . With benzene as the s o l -vent , the appearance o f i n t e r f e r r i n g peaks prevented the q u a n t i t a t i o n of the d e r i v a t i v e formed. (Th is was observed wi th the f lame i o n i z a t i o n de-t e c t o r G.L .C.) ( F i g . 3 ) . On the bas i s of maximal s e n s i t i v i t y and absence of any i n t e r f e r e n c e , HFBA was se l e c t ed as the d e r i v a t i z i n g agent of cho i c e . 2. S e l e c t i o n of an In te rna l Standard Quan t i t a t i on of compounds by G.L.C. ne ces s i t a t e s the use o f an i n t e r na l s tandard . An i dea l i n t e r n a l standard should chromatograph we l l under the cond i t i on s used and be we l l r eso lved from the compound to be q u n t i t a t e d . W-49167 (compound-I l l ) , a s t r u c t u r a l analog of t o ca i n i de was f i r s t s tud ied as the i n t e r n a l s tandard . Even though . the fo rmat ion of the hepta-f l u o r o b u t y r y l d e r i v a t i v e of W-49167 was complete w i t h i n 30 minutes, i ncon-s i s t e n t responses r e s u l t e d from occas iona l t a i l i n g of the peak and g l a ss adso rp t i on (F ig. 4A) . Add i t i o n of i sobutano l tended:to inc rease the response of the d e r i v a t i v e , p o s s i b l y by min im iz ing the adso rp t i on problems. The presence of an u n i d e n t i f i e d i n t e r f e r r i n g peak from b i o l o g i c a l samples made q u a n t i t a t i o n imposs i b l e . Add i t i on of water and ammonium hydroxide to remove the excess d e r i v a t i z i n g agent r e s u l t e d i n a d r a s t i c r educ t i on i n G.L.C. response, perhaps due to the i n s t a b i l i t y of the d e r i v a t i v e i n the presence of water . E f f o r t s to use monoethyl g l y c i n e xy l id ide,(compound IV) another analog o f t o c a i n i d e , a s an i n t e r n a l standard were a l so - 46 -unsuccess fu l , s ince m u l t i p l e peaks whose response changed w i th time were observed ( F i g . 4B) . The use of diazepam as a po s s i b l e i n t e r na l s tandard , r equ i r ed the a p p l i c a t i o n of temperature programming ( F i g . 4C) . I n i t i a l temperature: 180°; I n i t i a l t ime: 5 min. Rate: 30°/min. ; F i na l temperature: 260° Time of ho ld : 10 min. Retent ion time of Toca in ide: 4.3 min. Retent ion time o f Diazepam: 11.6 min. Column: 3% OV-17 Time o f a n a l y s i s : approx imate ly 17 min. Th is not on ly increased the time of a n a l y s i s but a l s o shortened the l i f e of the column. Wal le and coworkers [90] have repor ted the use of a-bromonaphthalene as an i n t e r n a l s tandard f o r the q u a n t i t a t i o n of a number o f amino compounds us ing d e r i v a t i z a t i o n procedures. a-Bromonaphthalene was found to s a t i s f y v i r t u a l l y a l l of the requirements o f a s u i t a b l e i n t e r n a l s tandard . I t gave a sharp peak which was we l l r e so l ved from the t o ca i n i d e d e r i v a t i v e peak and no i n t e r f e r r i n g peaks were observed. a-Bromonaphthalene a l s o showed a h i gh l y s a t i s f a c t o r y l i n e a r response over a range o f 900 pcg-3.8 ng per i n j e c t i o n . Hence, t h i s compound was chosen as the i n t e r na l standard f o r f u r t h e r s tud ies (1.8 ng/5 y l ) . 3. Op t im i za t i on of De r i v a t i v e Formation S ince the process o f d e r i v a t i z a t i o n i nvo l ves a chemical r e a c t i o n , i t i s necessary to op t im i ze the cond i t i ons of the r e a c t i o n . F igure 4: Gas chromatogram-rof d i f f e r e n t i n t e r n a l standards eva luated us ing a 2% OV-17 column. A. W-49167 (2ng) B. MEGX (4 ng) C. Diazepam (800 peg). - 48 -a) K i n e t i c s of De r i v a t i v e Formation The temperature and the r e a c t i o n time con t ro l the r a t e of d e r i v a -t i v e f o rmat i on . By ma in ta in ing the temperature constant a t 55°, the e f f e c t of d i f f e r e n t i n cuba t i on t ime on the y i e l d of the d e r i v a t i v e was moni tored. The t o ca i n i de base was prepared as mentioned i n Sec t i on I I . E . l . A f t e r d i s s o l v i n g the base i n hexane (1 m l ) , 10 u l of HFBA was added to the r e a c t i o n m ix tu re . Samples were incubated f o r d i f f e r e n t per iods of time (30, 40, 45, 50, 55, 60, 70, 80, 90 and 100 m i n . ) . The contents of the tubes were evaporated under a gent l e stream of n i t rogen and the res idue r e c on s t i t u t e d wi th 1 ml of i n t e r n a l standard s o l u t i o n . F ive y l o f t h i s s o l u t i o n was i n j e c t e d in to the G.L.C. and the response was monitored as the area r a t i o of the d e r i v a t i v e / i n t e r n a l standard ( F i g . 5 ) . b) S e l e c t i o n o f a So lvent f o r React ion Ch loro form, methylene c h l o r i d e , hexane and benzene were examined f o r s u i t a b i l i t y as so l ven ts f o r the d e r i v a t i z i n g r e a c t i o n . Toca in ide base was obta ined from the hydroch lo r i de s a l t as mentioned i n Sec t i on I I . E . l . Free base was then d i s s o l v ed e i t h e r i n 1 ml of ch lo ro fo rm, methylene c h l o r i d e , benzene or hexane and 10 u l o f HFBA was added to the s o l u t i o n . A f t e r vo r t ex i ng the con ten ts , the tubes were incubated f o r 50 minutes a t 55°. A f t e r t h i s t ime per iod the contents were coo led to room temperature and evaporated under n i t r ogen . The res idue was d i s s o l v ed i n 1 ml of the i n t e r na l s tandard s o l u t i o n and 5 u l of the sample was i n j e c t e d i n t o the G.L.C. The use of hexane as the so l ven t gave the maximum y i e l d o f the d e r i v a t i v e . A R E A RATIO, MONO-HEPTAFLUOROBUTYRYL DERIVATIVE OF 2 - A M I N 0 - 2 ' - 6 ' PROPIONOXYLIDIDE /oc - B R O M O N A P H -- T H A L E N E O O O O O O O - 617 - 50 -c) S e l e c t i o n o f the Volume o f the Reagent Used Depending upon the amount of the d e r i v a t i z i n g agent used f o r the r e a c t i o n , the r ea c t i on may or may not proceed to comple t ion . Experiments were c a r r i e d out us ing d i f f e r e n t volumes (5 , 10, 20, 30 y l ) of HFBA. Under the concen t ra t i ons o f t o c a i n i d e r o u t i n e l y ana lyzed , 10 y l was found to be s u f f i c i e n t f o r the complet ion o f the r e a c t i o n . 4. Removal of the Excess D e r i v a t i z i n g Agent The t o ca i n i d e base was d i s s o l v ed in hexane (1 ml) and 10 y l o f HFBA was added to the m ix tu re . The r e a c t i o n was a l lowed to proceed a t 55° f o r 50 minutes. The samples were a l lowed to cool to room temperature. One se t o f samples was evaporated to dryness under n i t rogen and r e c o n s t i t u t e d w i th 1 ml of i n t e r na l standard s o l u t i o n . To the other se t of tubes, 0.5 ml 'water was added and the s o l u t i o n was vo r texed . 0.5 ml o f 1% ammonium hydroxide was added to the tubes and the s o l u t i o n vortexed aga in . F ive y l o f the organ ic l a ye r was i n j e c t ed i n to G.L.C. and the responses were compared between two d i f f e r e n t methods of removing the excess reagent . 5. S t a b i l i t y o f the De r i v a t i v e Formed The t o ca i n i de d e r i v a t i v e formed a f t e r i n cuba t ing the f r ee base w i th 10 y l o f HFBA f o r 50 minutes a t 55° was a l lowed to stand at room temperature f o r va ry ing per iods of t ime and the response of the samples recorded a t d i f -f e r en t t ime i n t e r v a l s . The response as measured by the area r a t i o of the t o ca i n i de d e r i v a t i v e to the i n t e r n a l standard i s shown i n F igure 6. Add i t i o na l exper imentat ion c a r r i e d out on a long-term bas i s showed tha t the d e r i v a t i v e was s t ab l e f o r over a per iod of 4 days. (The area / r a t i o o f the d e r i v a t i v e / i n t e r n a l stand remained constant over t h i s pe r i od . ) 1— 1 1 1 I 2 3 4 TIME IN HOURS F igure 6: S t a b i l i t y of the d e r i v a t i v e formed, a t room temperature,as measured by the area r a t i o o f the d e r i v a t i v e / i n t e r n a l s tandard. Each data po in t i s the mean o f 4 ob se rva t i on s . - 52 -6. Conf i rmat ion of the S t ru c tu re of the De r i v a t i v e Formed Conf i rmat ion o f the s t r u c t u r e of the d e r i v a t i v e formed was made by the G.L.C.-mass spectrometry o f the d e r i v a t i v e formed. A F inn igan model 9500 automated e l e c t r o n impact (EI) G.L.C. mass spectrometer was used under the f o l l ow i n g c ond i t i o n s : G.L.C. Cond i t i ons : Column - 1.8 m x 2 mm ( i . d . ) g l ass column packed w i th 3% OV-17 on chromosorb W.H.P. (80-100 mesh) I n j e c t i o n temperature - 250°C Oven temperature - 180°C C a r r i e r gas (Helium) f low r a t e 25 ml/min. Mass Spectrometer Cond i t i ons : I o n i z a t i o n beam energy - 70 eV E l e c t r on m u l t i p l i e r vo l tage - 2 KV Ana lyse r temperature - 75°C Separate temperature - 180°C I n j e c t o r temperature - 180°C 7. Op t im i za t i on of G.L.C. Cond i t i ons a) Column S e l e c t i o n Toca in ide hydroch lo r i de s o l u t i o n (0.5 ml of a stock s o l u t i o n con-t a i n i n g 4.09 yg/ml) was p ipe t t ed i n to a 50 ml c en t r i f u g e tube. A f t e r the add i t i o n of 1 ml of IN sodium hydrox ide, the aqueous s o l u t i o n was ex t r a c t ed w i th 10 ml of methylene c h l o r i d e . A l i q u o t s o f 7 ml of the organ ic l a ye r were then t r a n s f e r r e d to a 15 ml c en t r i f u ge tube and evaporated to dryness under n i t r o gen . The res idue was d i s s o l v ed in 1 ml hexane and 10 y l of - 53 -HFBA was added to the hexane s o l u t i o n . The sample was incubated a t 55° f o r 50 minutes. A f t e r c oo l i ng to room termperature , the sample was evapor-ated to dryness under a gent l e stream o f n i t rogen and the res idue was d i s -so lved i n 2 ml o f i n t e r n a l standard s o l u t i o n . F ive y l samples were i n j e c t e d i n t o a number of chromatographic columns and the response noted. b) Op t im i za t i on of Some G.L.C. Parameters The c a r r i e r gas f low ra te and the temperature o f the oven, i n j e c t i o n port and the de tec to r i n f l u ence the response of a compound i n G.L.C. a n a l y s i s . i Using a f i x e d i n j e c t i o n port temperature (200°) and de tec to r temperature (300°), the temperature o f the oven and the c a r r i e r gas f low r a t e were manipulated to get optimum response. One ml of an aqueous stock s o l u t i o n con ta i n i ng 2.02 yg/ml was ex-t r a c t ed and d e r i v a t i z e d as mentioned i n Sec t i on I I . E . 5 . Keeping the oven temperature at 180°,the de tec to r temperature (300°) and the c a r r i e r gas f low ra te 40 m l /m in . , the response of the d e r i v a t i v e to changes i n the i n j e c t i o n por t temperature was monitored from 200° to 300° at 10° i n t e r v a l s . One-hal f a ml o f an aqueous s o l u t i o n o f t o c a i n i d e con ta i n i ng 2.02 yg/ml of the drug was ex t r a c t ed and d e r i v a t i z e d as mentioned i n Sec t i on I I . E . 4 . Th is s o l u t i o n was used to determine the e f f e c t o f de tec to r tem-perature on the response o f the d e r i v a t i v e formed using an i n j e c t i o n port temperature of 200°, oven temperature 180°, and c a r r i e r gas f low ra te 40 ml/min. - 54 -8. Op t im i za t i on of E x t r a c t i o n a) S e l e c t i o n of So lvent f o r E x t r a c t i o n One ml o f the stock s o l u t i o n con ta i n i ng 4.04 yg/ml o f t o c a i n i d e hydroch lo r ide was sepa ra te l y p i pe t t ed i n t o twenty- four 50 ml c en t r i f u ge tubes. A f t e r the a dd i t i o n of 1 ml T.N sodium hydroxide, e x t r a c t i o n s were c a r r i e d out i n quad rup l i c a te us ing 10 ml of methylene c h l o r i d e , e thy lene c h l o r i d e , ch lo ro fo rm, e the r , hexane or benzene. A f t e r shaking f o r 15 minutes, 7 ml o f the organ ic l a ye r was t r a n s f e r r e d to a 15 ml c en t r i f u ge tube and the s o l u t i o n evaporated to dryness under a gent le stream of n i t r ogen . The res idue i n the tubes was d i s s o l v ed i n 1 ml hexane and d e r i v a t i z e d as mentioned e a r l i e r (Sec t ion 11.E .3-b). The response of the d e r i v a t i v e formed as measured by the area r a t i o o f the d e r i v a t i v e / i n t e r n a l standard was compared to determine the so l ven t w i th opt imal e x t r a c t i o n p r o p e r t i e s . b) Phase-Volume Rat io E x t r a c t i o n s of the aqueous s o l u t i o n of t o ca i n i de were a l s o c a r r i e d out us ing d i f f e r e n t phase volume r a t i o s (1 :1 , 1:2, 1:3, 1:4, 1:5) of the aqueous to organ ic l a ye r .withy methylene c h l o r i d e as the s o l v en t . A 1:5 aqueous to organ ic l a ye r r a t i o was found to g ive the maximum e x t r a c t i o n and was used i n l a t e r a n a l y s i s . c) M u l t i p l e E x t r a c t i o n vs S i ng l e E x t r a c t i o n Experiments were performed to determine the e f f e c t of s i ng l e /doub l e / t r i p l e e x t r a c t i o n w i th methylene c h l o r i d e . The use of 1:5 aqueous to organ ic phase volume r a t i o y i e l d e d r e cove r i e s comparable to double and t r i p l e e x t r a c t i o n us ing 1:2 aqueous to organ ic phase r a t i o . - 55 -I I . F. RECOVERY STUDIES 1. Standard Curve of Toca in ide Base About 2 mg of the t o ca i n i de base prepared as desc r ibed in Sec t i on I I . C . l was d i s s o l v ed i n 250 ml o f methylene c h l o r i d e and a s e r i e s o f s o l u -t i on s (6) having the f o l l ow i ng concen t ra t i on was prepared (60 ng/ml-600. ng/ml) . The methylene c h l o r i d e was evaporated to dryness and the res idue d i s s o l v ed i n 1 ml of hexane. Ten y l - o f HFBA. was added to the mixture arid the contents vo r texed . The samples were then incubated i n an oven a t 55° f o r 50 minutes. A f t e r complet ion of the r e a c t i o n , the samples were a l lowed to cool to room temperature (10 min.) and then evaporated to dryness under n i t rogen a t room temperature (25-30 m i n . ) . One ml of the i n t e r na l standard s o l u t i o n was added and the contents mixed wel l by v o r t e x i n g . F ive y l o f the sample was i n j e c t ed in to the G.L.C. and the response was noted. Standard curves were prepared by p l o t t i n g the amount of t o c a i n i d e i n j e c t ed in to the G.L.C. v£ the response r a t i o of the d e r i v a t i v e / i n t e r n a l s tandard . 2. Standard Curve f o r the Plasma/Urine E x t r a c t An aqueous s o l u t i o n of t o c a i n i d e hydroch lo r i de (0.1 to 0.6 ml i n 0.1 ml increments) was added to 0.1 ml of blank p lasma/ur ine . S u f f i c i e n t water was then added to make up to a volume of 1 m l . One ml of IN; sodium hydro-x ide was added and the s o l u t i o n s were shaken wi th a w r i s t a c t i on s h a k e r 4 1 f o r 15 minutes a f t e r the add i t i o n of 10 ml of methylene c h l o r i d e . Seven ml of the methylene c h l o r i d e l a ye r from each tube was t r a n s f e r r e d to 6 d i f f e r e n t 15 ml c en t r i f u ge tubes^and evaporated to dryness B u r r e l l w r i s t a c t i o n shaker, B u r r e l l Co rpo ra t i on , P i t t s b u r g h , Pennsy lvan ia . - 56 -us ing a gen t l e stream of n i t r ogen . The res idue was d i s s o l v ed in 1 ml of hexane and the contents of the tube d e r i v a t i z e d wi th HFBA as mentioned i n the prev ious sec t i on ( I I . E - 3 b ) . The d e r i v a t i v e s of t o c a i n i d e formed were r e con s t i t u t ed i n 2 ml of i n t e r na l standard s o l u t i o n . F ive y l o f the s o l u -t i o n was i n j e c t ed i n to the G.L.C. and the response r a t i o of the- d e r i v a t i v e / i n t e r na l standard was c a l c u l a t e d . Standard curves were prepared by p l o t t i n g the amount i n j e c t ed aga ins t the response r a t i o observed. Recovery o f t o c a i n i d e by the e x t r a c t i o n procedure was c a l c u l a t e d by comparing the expected response (from f r e e base standard curve) and the observed response (p lasma/ur ine e x t r a c t ) . I I . G. FAECAL EXCRETION STUDIES Two d i f f e r e n t doses (10 mg/kg and 20 mg/kg) of t o ca i n i de were ad-m in i s t e red to r a t s (2) i n t r a a r t e r i a l l y v i a the t a i l a r t e r y . ; 24 hour faeca l samples were c o l l e c t e d by means of a s p e c i a l l y designed screen at tached to the metabo l i c cages. Faeces samples were a l s o c o l l e c t e d from animals i n j e c t ed w i th 0.5 ml s a l i n e . The faeca l samples ( a i r d r i ed ) were weighed and homogenized i n a l i q u o t s of d i s t i l l e d water. 0.5-1 ml of t h i s homogenate was analyzed f o r the presence of i n t a c t t o c a i n i d e . The general a n a l y t i c a l procedure was the same as tha t used f o r the a n a l y s i s o f u r ine and plasma. The d e r i v a t i z e d samples were analysed by both F . I .D . -G .L .C . and E . CD . -G . L . C . methods. Fur ther exper imentat ion was c a r r i e d out to ana lyze f o r the presence of any conjugated t o ca i n i d e i n the faeca l samples. 1 ml of the faeca l homogenate was subjected to a c i d h yd r o l y s i s ( incubate a t 100° f o r 1 hr . - 57 -w i th 1 ml T.N HC1 i n a sea led ampoule). The a c i d hydro lyzed samples were ex t rac ted by the general procedure desc r ibed i n scheme 1 . I I . H. STABILITY OF TOCAINIDE AT 55°C Stud ies were c a r r i e d out a t 55°C to determine the a b i l i t y o f t o ca i n i de to w i ths tand t h i s temperature dur ing the process of d e r i v a t i z a -t i o n . One ml of t o ca i n i de hydroch lo r i de (4.04 yg/ml) was ex t rac ted w i th 10 ml of methylene c h l o r i d e a f t e r the a d d i t i o n of 1 ml IN sodium hydrox ide . A l i q u o t s of 7 ml of the methylene c h l o r i d e l a ye r was then t r an s f e r r ed to 15 ml c en t r i f u ge tube and evaporated to dryness under n i t r ogen . The res idue was r e c on s t i t u t e d in 1 ml hexane and the c en t r i f u ge tube was incubated a t 55°C f o r 50 minutes. A f t e r t h i s t ime pe r i od , the contents of the tube were coo led to room temperature and evaporated to d ryness . One hundred y l o f a benzene s o l u t i o n of a-Bromonaphthalene ( i n t e r na l s tandard 0.36 ng/y l ) was added to the c en t r i f u ge tube to d i s s o l v e the r e s i due . -Three y l of t h i s s o l u t i o n was then i n j e c t e d in to F . I .D . -G . L .C . The response o f t h i s sample was compared to another sample which was prepared i n a s i m i l a r f ash ion but w i thout any i n cuba t i on a t 55° f o r 50 minutes. Greater than 95% of the o r i g i n a l amount incubated was present f o l l ow i n g 50 minute i n cu -bat ion a t 55°.• Th is suggests the absence of a n y . s i g n i f i c a n t degradat ion" o f t o c a i n i d e a t 55° i n hexane s o l u t i o n . - 58 -I I . I . OPTIMIZED ANALYTICAL CONDITIONS The f i n a l e x t r a c t i o n procedure chosen f o r subsequent a n a l y s i s i s shown in the accompanying f low cha r t , (scheme 1) The f o l l ow i n g G.L.C. c ond i t i on s were used f o r the a n a l y s i s o f t o ca i n i de in b i o l o g i c a l f l u i d s . Instrument Hewlett Packard Model 5833-A r epo r t i n g gas chromatograph equipped 6 3 wi th N i - e l e c t r o n capture d e t e c t o r . Column A 1.8 m :x 2 mm ( i . d . ) g l a s s column con ta i n i ng 3% OV-17 coated on 80-100 mesh chromosorb W(H.P.), A l l columns were cond i t i oned as f o l l ow s : 24 hours a t 100°' ; then the temperature was increased to 280° a t the ra te of 0.'l°/min. The columns were maintained, a t 280° f o r a f u r t h e r 24 hour pe r i o d . The c a r r i e r gas f low ra te dur ing the c ond i t i o n i ng per iod was 7 ml/minute. Hi-Temp vespel® f e r r u l e s ^ ? were used to plumb the column. Microsep® gas chromatography s e p t a ^ were used f o r the i n j e c t i o n po r t . Operat ing Cond i t i ons Temperature: I n j e c t i o n port 200° Oven 180° Detector 300° A l l t e c h A s so c i a t e s , A r l i n g t o n He igh ts , I l l i n o i s . Un imetr i cs Un ive rsa l Co rpo ra t i on , Anaheim, C a l i f o r n i a . - 59 -- Scheme 1 E .C.D.-G.L .C. Ana l y s i s of Toca in ide Blood Cent r i f uge Plasma ) 100 y l > Sodium hydroxide 1 ml Ur ine 100-200 y l Cent r i fuge Tube Shaken f o r 15 min, Cent r i fuged to sepa-r a t e . 1ayers 7 ml M.C. l a ye r H 20 q . s . 2 ml Methylene Ch lo r i de 10 ml 10 y l H.F.B.A. Evaporate D i s so l ve res idue i n 1 ml Hexane React ion a l lowed to proceed at 55°C f o r 50 min. Evaporat ion 1 Disso l ve res idue i n 100-1000 y l Benzene con ta in i ng i n t e r na l standard (approx. 1.8 ng/5 y l ) 1-5 y l i n j e c t ed i n G.L.C. (E.C.D.) - 60 -C a r r i e r gas (95% Argon-5% Methane) .,40":ml/min. Isothermal run was used Time o f a n a l y s i s : 10-12 min. Order of emergence of peaks: Retent ion t ime Compound a-Bromonaphthalene ( i n t e r n a l standard) Toca in ide d e r i v a t i v e 1.96-2.00 min. 4.30-4.50 min. Quan t i t a t i v e Ana lyses Responses of the samples were est imated as the area r a t i o of the t o ca i n i de d e r i v a t i v e / i n t e r n a l s tandard . Range of standard curve: 0.2 ng to 3.2 n g / i n j e c t i o n . I I . J . DRUGS, DOSES AND DOSAGE FORMS A stock s o l u t i o n con ta i n i ng 9.52 mg/ml of t o c a i n i d e hydroch lo r i de (corresponding to 8.00 mg/ml of t o c a i n i d e base) in normal s a l i n e or d i s t i l l e d water was prepared. Th is s o l u t i o n was s u i t a b l y d i l u t e d to g i ve s o l u t i o n s of the f o l l o w i n g concen t ra t i on (8 mg/ml, 6 mg/ml, 4 mg/ml,.2 mg/ml.of base) 0.5 m l ' o f these s o l u t i o n s g iven to the r a t corresponds to 20 mg/kg (8 mg/ml), 15 mg/kg (6 mg/ml), 10 mg/kg (4 mg/ml), and 5 mg/kg (2 mg/ml). So l u t i on s con ta in i ng 19.041 mg/ml of t o c a i n i d e hydroch lo r i de (0.5 ml = 40 mg/ml), were a l s o prepared. Th is was s u i t a b l y d i l u t e d to g ive s o l u t i on s 12 mg/ml (30 mg/kg). arid 10 mg/ml (25 mg/kg). - 61 -Phenobarb i ta l Sodium A 280 mg sample of phenobarb i ta l sodium was d i s s o l v ed in 10 ml normal s a l i n e . One-hal f ml of t h i s s o l u t i o n was admin i s te red to the r a t (dose = 70 mg/kg) i n t r a p e r i t o n e a l l y . SKF 525-A About 200 mg of SKF 525-A weighed a c cu r a t e l y was d i s s o l v ed i n 10 ml of normal s a l i n e and 0.5 ml of t h i s s o l u t i o n was admin is tered to the r a t s (dose 50 mg/kg) i n t r a p e r i t o n e a l l y . Sal i cy!amide A 2 gm sample of s a l i c y l am i de was d i s s o l v ed i n minimal amount of sodium hydroxide and then .ad jus ted to pH" 9 .7-10, by the a d d i t i o n of d i l u t e hyd roch l o r i c wh i l e s t i r r i n g . The s o l u t i o n was then made up to 25 ml w i th d i s t i l l e d water . Sa l i c y l am ide s o l u t i o n s were f r e s h l y prepared p r i o r to use. One-hal f ml of the above s o l u t i o n , was admin is tered to the r a t s ' dose: 200 mg/kg) e i t h e r i n t r a p e r i t o n e a l l y or o r a l l y . Table VII summarizes the d i f f e r e n t treatment and pretreatment s,c schedules employed i n t h i s s tudy. U . K . ANIMAL STUDY Adu l t male Wis tar r a t s w i th an average weight of 200 gms (190-210 gms) were used in a l l experiments (animals were obta ined from the UBC animal care u n i t ) . The animals were mainta ined in m e t a l l i c cages (41 cm x 34-cm x 18 cm) (6 to 8 per cage) i n a c o n t r o l l e d environment (temperature -4°, humidi ty - dry) f o r a t l e a s t three days p r i o r to the exper iments . - 62 -Table VII Treatment - Pretreatment Schedule Experiment Bas i c K i n e t i c s Pretreatment Drug and Dose Route of Admin i -s t r a t i o n Route of Admin i -s t r a t i o n of Toca in ide i . v . Oral i . p . Dose of Toca in ide (mg/kg) 5 ,10,15,20, 25 , 30 , 40 a 5,10,15,20° 5,10,15,20° Phenobarb i ta l Study A) a) con t ro l b) Test B) a) con t ro l b) Test 0.5 ml s a l i n e f o r 3 days p r i o r to the experiment i . p . 0.5 ml Phenobar-b i t a l sodium (70 mg/kg) f o r three days p r i o r to the experiment i . p . 0.5 ml s a l i n e f o r 7 days p r i o r to the experiment i . p . 0.5 ml Phenobar-b i t a l sodium (70 mg/kg) f o r 7 days p r i o r to the experiment i . p . Oral Oral Oral i . v . Oral i . v . 20 L 20" 20' 20 c 20° 2 0 a SKF 525-A A) a) con t ro l b) Test 0.5 ml s a l i n e 40 min. p r i o r to the adm in i s t r a t i on of t o ca i n i d e i . p . 0.5 ml SKF 525-A 40 min. p r i o r to the adm in i s t r a t i on of t o c a i n i d e i . p . 1 . v . Oral i . v . Oral 1 5 a 2 0 c 15 , 20 c 15° 2 0 c 15,20° - 63 -Table VII ( cont 'd ) 4. Sa l i c y l am ide A) a) con t ro l b) Test B) a) con t ro l b) Test C) a) con t ro l b) Test NaOH/HCl s o l u t i o n to « pH 10.0 s imu l -taneous ly w i th t o ca i n i d e i . p . Oral 15 Sa l i c y l am ide (200 mg/kg) in sodium hydroxide [pi! 9 .7-10.0] i . p . Oral 15 c NaOH/HCl s o l u t i o n « pH 10.0 given 10 min. p r i o r to adm in i s t r a t i on of t o c a i n i d e Oral Oral 1 5 c Sa l i c y l am ide (200 mg/kg) in sodium hydroxide (pH 9.7-10.0) Oral Oral 15 NaOH/HCl s o l u t i o n pH « 10.0 30 min. p r i o r to admin i -s t r a t i o n of t o c a i n i d e i . p . Oral 1 5 c Sa l i c y l am ide (200 mg/kg) in sodium hydroxide pH (9 .7 -10.0) 30 min. p r i o r to adm in i s t r a t i o n of t o c a i n i d e i . p . Oral 1 5 c a u r i ne and plasma l e v e l study L. plasma l e ve l study on ly c u r i ne l e v e l study on ly - 64 -Wooden shavings were used as the bedding under e levated cages (18 cm from the bottom of the cage) . The photo-per iod was c o n t r o l l e d to prov ide dark from 20.00 hours to 06.00 hours and l i g h t from 06.00 to 20.00 hours. Rat chow or lab chow was fed to the an ima l s . The animals were f a s t ed f o r a per iod of 8-10 hours p r i o r to and dur ing the exper iments. Water was a l lowed ad_ 1 ib i turn. 1. Plasma Level Study The plasma l e ve l study of t o c a i n i d e was c a r r i e d out i n animals w i th an implanted j u g u l a r ve in cannu la . The d e t a i l s of the cannu la t i on procedure are as f o l l o w s . The can-nu l a t i on procedure i s a minor mod i f i c a t i o n of the technique developed by Weeks and coworkers [91 ] . a) P repara t i on of the Cannula The j u g u l a r ve in cannula c on s i s t s o f po lye thy lene tub ing (PE 50) j o i ned to s i l a s t i c tub ing ( s i l a s t i c medical grade) by means of a shor t length of hypodermic needle t ub i ng . A p iece of PE 50 tub ing (15 cm) i s f l a r e d a t one end w i th a b l un t 22 gauge needle and the 4 mm length o f hypo-dermic needle tub ing (22 gauge) i s i n se r t ed to a d i s tance of about 2 mm. The s i l a s t i c tub ing (3-4 mm) i s threaded from the exposed end of the needle tub ing and pos i t i oned over the se c t i on of po lye thy lene covered needle t ub i ng . A p iece of 4-0 s i l k thread i s used to secure the t ub i ng . The cannula end o f the po lye thy lene tub ing i s formed i n t o a "U" by ; bending around a pasteur p i p e t t e and d ipp ing momentari ly i n to b o i l i n g water . A 180° bend i n a plane perpend i cu la r to the "U" i s made on the PE 50 t ub i ng , - 65 -near l y 3-4 mm away from the cannula end, thus making the cannular conform to i t s p o s i t i o n i n the r a t . ( F i g . 7) The cannula i s c leaned w i th d i s t i l l e d water , soaked in 95% a l coho l f o r 3-5 hours and s to red in s t e r i l e normal s a l i n e s o l u t i o n p r i o r to use. b) Su rg i ca l Procedure The r a t i s anaes the t i zed w i th ether and the ha i r removed from the back of the neck and over the r i g h t j u gu l a r v e i n . The area i s c leaned wi th su rg i c a l soap. A t ransverse i n c i s i o n i s made i n the sk i n s l i g h t l y behind the shoulder blade and the area around the i n c i s i o n i s debr ided of f a s c i a w i th f o r ceps . The animal i s then turned on i t s back and a l o ng i t u d i n a l i n c i s i o n i s made over the r i g h t ex te rna l j u g u l a r v e i n . A few mm of the ve in i s exposed by b lun t d i s s e c t i o n . The ve in i s l i g a t e d a t the upper end us ing a s i l k su tu re . A probe i s i n s e r t ed subcutaneously j u s t behind the ear , from 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 . The cannu la , f i l l e d w i th hepar in i zed s a l i n e (20 U/ml),, i s then i n s e r t ed i n t o the probe and the probe i s pu l l ed towards the j ugu l a r v e i n . I f the cannula, is:>bent.; i n the c o r r e c t p lane, the U po r t i on w i l l l i e f l a t on the neck muscle. A probe i s then p laced under the ve in and a small puncture i s made in to the ve in w a l l . The t i p o f the s i l a s t i c tub ing i s cu t i n to a 45° bevel so as to f a c i l i t a t e easy i n s e r t i o n of the tube i n to the v e i n . The tube i s then advanced s l ow ly up to the j u n c t i o n o f the s i l a s t i c - p o l y e t h y l e n e t ub i ng . I t i s secured i n the ve in w i th 4 - 0 . s i l k su tu re . The cannula i s anchored i n p o s i t i o n by t y i n g the thread (used to secure the tub ing i n the ve in ) to the adjacent muscle ( s ca l enus ) . - 66 -F igure 7: Cannula used f o r j u g u l a r ve in cannu la t i on - 67 -The subcutaneous connect ive t i s s u e i s r eun i t ed w i th s u r g i c a l thread to cover the PE tub ing and the sk i n i n c i s i o n i s c losed us ing a 0-0 s i l k . One end of the cannula protrudes through the i n c i s i o n on the back of the neck. The cannula i s secured i n p o s i t i o n by c l o s i n g the s k i n i n c i s i o n w i th a 0-0 s i l k . The cannula i s checked to ensure tha t i t i s f u n c t i o n a l . About 4 cm of the cannula i s a l lowed to protrude from the nape of the neck; the cannula i s sea led us ing a p i n . P e r i o d i c use (on a l t e r n a t e days) of the hepar in i zed s a l i n e (20 U/ml) helped to keep the cannula f u n c t i o n a l . The animals recover w i t h i n minutes of the surgery . However, a m i n i -mum per iod o f th ree days was a l lowed f o r complete recovery . In experiments i n v o l v i n g phenobarb i ta l pret reatment , the surgery was done on the 495th day o f the pretreatment schedu le . c) Exper imentat ion Plasma l e v e l s tud i e s were c a r r i e d out a f t e r in t ravenous adm in i s t r a -t i o n of d i f f e r e n t doses of t o c a i n i d e . About 0.2 ml of hepar in i zed s a l i n e (20 U/ml) was taken i n a 1 ml s y r i n g e ^ w i th a 23 G needle^ 5 and 0.2 ml of blood was withdrawn from the animal v i a the j u gu l a r ve i n cannu la . One-hal f ml of an aqueous s o l u t i o n ( i n normal s a l i n e ) of t o c a i n i d e hyd roch l o r i de , c o r -responding to 5, 10, 15, 20 and 40 mg/kg (expressed as base) was i n j e c t e d through the j u gu l a r ve in cannu la . The cannula was then f l u shed With the contents of the f i r s t sy r inge (0.2 ml blood + 0.2 ml hepar in i zed s a l i n e ) . Thus, the r a t was ensured of r e c e i v i n g the e n t i r e dose. Glaspak 1 c c . t u b e r c u l i n syr inge/B-D y a l e t u b e r c u l i n 1 c c . g l ass s y r i n ge . Beckton, D ick inson and Co. , Ru the r fo rd , N.J . Ya le needle (Luer lok hub) s t a i n l e s s cannula 23G1. Beckton, D ick inson and Co. Canada L t d . , M i s s i s sauga , On ta r i o . - 68 -At app rop r i a t e time i n t e r v a l s (10, 20, 30, 60, 90, 120, 150, 180, 240, 300, 360, 480, 600 min.) a sy r i nge w i th 0.2 ml of hepar in i zed s a l i n e was in t roduced i n to the cannula and about 0.15 ml of the f l u i d was w i t h -drawn. A f r e s h needle and syr inge was in t roduced and a 100-250 u l blood sample was removed from the an ima l . The s o l u t i o n in the syr inge (contents from the cannu la , some blood and the hepar in i zed s a l i n e ) was i n j e c t e d back i n to the an ima l . AC The blood samples were t r a n s f e r r e d to a hepar in i zed caraway t u b e H D and one end was sea led using a cr itocap®.^ The tubes were cen t r i f uged f o r 10-15 minutes a t a speed 6 i n a 48 c l i n i c a l c en t r i f u ge to separate the plasma and the samples were f rozen immediate ly and kept f rozen u n t i l ana l yzed . 2. U r ina ry Exc r e t i on S tud ies For the u r i na r y e x c r e t i o n s t ud i e s , the drug was given o r a l l y ( g a s t r i c i n t u b a t i o n ) , i n t r a p e r i t o n e a l l y or i n t r avenous l y ( t a i l a r t e r y ) , a f t e r anaes-t h e t i s i n g the animal w i th e t h e r . 4 9 Dosed animals were housed i n separate s t a i n l e s s s tee l cages (24.5 cm x 17.5 cm x 18 cm) wi th f a c i l i t i e s f o r c o l l e c t i n g u r i ne samples, f r ee of any faeca l contaminat ion , i n an amber co loured b o t t l e . A f t e r 24 and 48 hours pos t -dos i ng , the s i des o f the cage and c o l l e c t i n g tubes-were washed w i th d i s t i l l e d water t h r i c e to recover a l l the exc re to ry products . '4^ Dade-caraway tube. 4 7 C r i t o caps ® (Regular) Sherwood Medical I n d u s t r i e s , S t . L ou i s , M i s s o u r i . 48 Damon/IEC D i v i s i o n , Needham H t s . , Massachuset ts . 4 9 Ether s o l ven t , U.S.P. - 69 -I I . L. DETERMINATION OF THE PRESENCE OF A TOCAINIDE CONJUGATE 1. A c i d Hyd ro l y s i s A 1 ml sample of u r i ne was p i pe t t ed i n t o a 5 ml g lass ampoule and 1 ml o f IN hyd ro ch l o r i c a c i d was added. To another 1 ml of u r ine sample taken i n another 5 ml g l a s s ampoule 1 ml of d i s t i l l e d water was added. Both ampoules were f l u shed w i th n i t rogen f o r few seconds and s ea l ed . The sea led ampoules were incubated a t 100° f o r a one hour p e r i o d . A f t e r the h yd r o l y s i s 0.2 ml sample was taken from both ampoules and analyzed f o r the i n t a c t t o c a i n i d e as desc r ibed e a r l i e r . 2. g l u c u r o n i d a s e Hyd ro l y s i s A f i v e ml sample oT ur ine was ad justed to, pH 5.5 us ing d i l u t e hydro-c h l o r i c a c i d and made up to a volume of 10 ml w i th d i s t i l l e d water . One ml of the above s o l u t i o n together w i th 0.2 ml of glucurase® and-0.3 ml of d i s t i l l e d water were t r a n s f e r r e d to a s c i n t i l l a t i o n v i a l . Contro l e x p e r i -ments were a l s o run i n the absence o f any enzyme. The v i a l s were incubated a t 37° i n a water bath f o r 24 hours. At the end of the i ncuba t i on pe r i od , the samples were analyzed f o r the amount.of i n t a c t t o c a i n i d e present . 3. E f f e c t of 1:4 Saccharo-Lactone (0.1 mM) on the Enzymatic Hyd ro l y s i s of Toca in ide Conjugate P repa ra t i on of the stock s o l u t i o n of 1:4 saccharo- l ac tone: A stock s o l u t i o n of 1:4 saccharo- lac tone was prepared by d i s s o l v i n g 2 mg of the compound i n 100 ml of ace ta te bu f f e r (pH 5 .0 ) . One ml of u r ine sample was added to each of 9 s c i n t i l l a t i o n v i a l s . To 6 v i a l s , ! ml o f ace ta te bu f f e r was added. To the other three v i a l s - 70 -1 ml of the stock s o l u t i o n of 1:4 saccharo- lac tone was added. Exa c t l y 0.20 ml of the enzyme s o l u t i o n (glucurase®) was added to three v i a l s w i th 1:4 saccharo- lac tone and three v i a l s w i thout any 1:4 saccharo l a c t one . The remaining v i a l s , to which 0.20 ml of water was added, were used as the c o n t r o l . 4. Op t im i za t i on of Enzyme Hyd ro l y s i s a) E f f e c t o f Enzyme (glucurase® ) Concent ra t ion on the Hyd ro l y s i s o f the Glucuron ide Conjugate Ur ine samples (5 ml) obta ined from ra t s dosed w i th t o c a i n i d e (15 . mg/kg) were ad jus ted to pH 5.5 us ing d i l u t e hyd roch l o r i c a c i d . The sample was then made up to 10 ml w i th d i s t i l l e d water i n a vo lumet r i c f l a s k . One ml of the sample was then p laced i n 9 s c i n t i l l a t i o n v i a l s . Vary ing volumes of the enzyme s o l u t i o n (0.10 through 0.40 w i th 0.10 ml increments) was then added to the v i a l i n dup l i c a t e (1-8 tubes ) . One tube w i thout any added enzyme served as the c o n t r o l . The requ i red amounts o f d i s t i l l e d water were added to b r ing the t o t a l volume i n each v i a l to 1.50 m l . The v i a l s were then capped and incubated a t 37°;,for .'a 24 hour time per iod i n a water ba th . At the end of the i ncuba t i on pe r i od , 0.50 ml o f the s o l u t i o n was ana lyzed f o r t o c a i n i d e , by the method descr ibed e a r l i e r . b) Comparison of the E f f e c t o f Acetate Bu f f e r .vs the use of D i l u t e Hydro-c h l o r i c Ac i d to Ad jus t the pH of the Incubat ion Medium P r e l im i na r y experiments were c a r r i e d out to compare the e f f e c t o f the ace ta te b u f f e r , and the IN hyd roch lo r i c a c i d used to ad jus t the.pH o f the , i ncuba t i on medium, on the hyd r o l y s i s o f the conjugates of t o c a i n i d e . Two ml of the u r ine sample was adjusted to pH 5.50 w i th hyd roch l o r i c a c i d and made up to a volume of 5 ml w i th d i s t i l l e d water ( s o l u t i o n A ) . To another - 71 -set o f 2 ml u r ine samples, 3 ml o f ace ta te bu f f e r (pH 5.50) was added ( s o l u t i o n B ) . One ml each of s o l u t i o n s A and B were t r a n s f e r r e d to four d i f f e r e n t s c i n t i l l a t i o n v i a l s . A l i q u o t s o f .the 0.2 ml enzyme s o l u t i o n (glucurase®) and 0.3 ml of d i s t i l l e d water were added to two tubes con-t a i n i n g s o l u t i o n A and two tubes con ta i n i ng s o l u t i o n B. The r e s t o f the four tubes to which 0.5 ml o f d i s t i l l e d water was added, acted as the con-t r o l s (w i thout any enzyme). A l l tubes were t i g h t l y capped and incubated a t 37° i n a water bath over a per iod of 24 hours. A f t e r the end of the i n c u -ba t ion pe r i od , a l i q u o t s o f the samples were ana lyzed f o r i n t a c t t o c a i n i d e . c) E f f e c t o f the Source of 3G1ucuronidase on the Hyd ro l y s i s o f the Conjugate of Toca in ide In the present s tudy, three d i f f e r e n t source of B-g lucuron idase enzymes ( g l u su l a s e , g lucurase and 3-g lucuron idase (mo l lusk)) were used to study t h e i r a b i l i t y to hydro lyse the conjugate of t o c a i n i d e . i ) G lusu lase vs_G lucu rase , - To a 1 ml u r ine sample p i pe t t ed in to each of 9 s c i n t i l l a t i o n v i a l s , 1 ml o f ace ta te bu f f e r (pH 5.0) was added. A l i q u o t s o f 0.2 ml of g lucurase were added to three v i a l s . G lusu lase (0.1 ml) and 0.1 ml of d i s t i l l e d water were added to .an add i t i o na l three v i a l s and to the remaining three v i a l s , 0 . 2 ml of d i s t i l l e d water was added. A l l of the v i a l s were then incubated a t 37°:' f o r 24 hours. A f t e r the incuba t i on pe r i od , a l i q u o t s of samples were analyzed f o r the presence of i n t a c t t o c a i n i d e . i i ) G lucurase vs^ 3-G1 ucuronidase (mol lusk) - A stock s o l u t i o n of mol lusk 3-Glucuronidase was prepared by d i s s o l v i n g 20 mg o f enzyme i n 10 ml of ace ta te bu f f e r pH 5.00. A 1 ml u r i ne sample was added to each o f 4 s c i n t i l l a t i o n v i a l s . To 2 v i a l s , 1 ml of a ace ta te bu f f e r (pH 5.0) was added, to the other 2 v i a l s - 72 -1 ml of the bu f fe red enzyme s o l u t i o n was added and the v i a l s were i n c u -bated as desc r ibed e a r l i e r . A l i q u o t s of the samples were ana lyzed f o r i n t a c t t o c a i n i d e . d) E f f e c t of Chloroform on the Enzymatic Hyd ro l y s i s of Toca in ide Conjugate Add i t i o na l exper imentat ion was a l s o c a r r i e d out to determine the e f f e c t of pH of the incuba t i on medium, the time of i n cuba t i on and the e f f e c t o f a c t i v a t o r s (ch loroform) on the enzyme mediated h y d r o l y s i s . 5. Op t im i za t i on of Ac i d Hyd ro l y s i s a) Incubat ion w i th Hydroch lo r i c A c i d i n an Oven a t 100° E f f e c t o f d i f f e r e n t s t rengths of a c i d and d i f f e r e n t t imes o f incuba-t i on- on the h y d r o l y s i s : - A l i q u o t s of 1 - ml each of u r ine were p i pe t t ed i n t o a c lean 5 ml g l a s s ampoule. One ml of e i t h e r water , TN, 2N, 6N or concen-t r a t ed hyd roch l o r i c a c i d was added to the ur ine sample. The ampoules were f l u shed w i th n i t r ogen f o r a per iod of 30 seconds and sea l ed . The sea led ampoules were then incubated i n an oven a t 100°.for per iods rang ing from 1, 2, 3, 4 to 6 hours i n d i f f e r e n t s tud ies to determine the e f f e c t of time of i n cuba t i on on the h y d r o l y s i s . b) Hyd ro l y s i s by Au toc l av ing w i th Ac i d In one exper iment, the ampoules prepared as mentioned in the prev ious sec t i on (sample + water; sample + IN ac i d ) were autoc laved a t 121° f o r . 35 minutes. The r e s u l t s obta ined by t h i s method were compared w i th the other method (1 hour a t 100°). - 73 -6. S t a b i l i t y o f Toca in ide Hydroch lo r ide So l u t i o n a t 100° Experiments were c a r r i e d out to determine the s t a b i l i t y of t o ca i n i de hydroch lo r i de s o l u t i o n a t 100° i n an oven i n the presence o f added water , I N , 2 N, 12 N hyd roch l o r i c a c i d and 12 N hyd roch l o r i c a c i d sp iked w i th b l a n k , u r i n e . 7. S t a b i l i t y of an Aqueous So l u t i o n of Toca in ide i n the Presence of Added Urea To 1 ml of an aqueous s o l u t i o n of t o c a i n i d e hydroch lo r i de (4.04 yg /m l ) , taken i n an ampoule,1 ml of 8 M urea s o l u t i o n was added. The ampoule was f l u shed wi th n i t rogen and s ea l ed . Th is sample along w i th a c o n t r o l , which conta ined 1 ml of the aqueous s o l u t i o n o f t o ca i n i de hydroch lo r i de and 1 ml of d i s t i l l e d water f l u shed and sea led i n a manner s i m i l a r to the t e s t ampoule, was incubated i n an oven a t 100° f o r 1 hour. A f t e r the i ncuba t i on t ime, the contents were ana lyzed f o r the amount o f i n t a c t t o c a i n i d e present . I I . M. M u l t i p l e Ana ly t i ca l -Scheme f o r Determining -the O r i g i n and Nature of the C y c l i c Me tabo l i t e 1. E x t r a c t i o n Procedure A m u l t i p l e a n a l y t i c a l pathway as descr ibed in scheme 2 was used to determine the presence of a c y c l i c metabo l i t e i n r a t u r i ne a f t e r t o c a i n i d e a d m i n i s t r a t i o n . Experiments designed helped in c l a r i f y i n g whether t h i s c y c l i c compound was i n f a c t a metabo l i t e or a ' 'metabonate". Rats were admin i s te red m u l t i p l e doses (2-3) of t o c a i n i d e ( app rox i -mately 40 mg/kg) g iven e i t h e r o r a l l y or i n t r a p e r i t o n e a l l y . The u r ine samples Scheme 2. Mu l t i p l e Ana l y t i ca l Scheme fo r Determining the Or i g i n and Nature of the Cyc l i c Metabol i te URINE SAMPLE (Tocainide/Deuterated toca in ide) © To pH 9.0 © With sodium hydroxide With ammonium ca r -bonate and ammonium hydroxide Mu l t i p l e Ex t rac t i on with methylene ch l o r i de Organic Layer Con-centrate a) b) Aqueous Layer adjust pH to 12.5 with sodium hydroxide and hold f o r 1-1.5 hrs br ing down to pH to 9.0 with d i l u t e hydro-c h l o r i c ac id 1 © Direct ex t rac t i on of ur ine sample (no pH ad jus t -ments) Mu l t i p l e Ext rac t ions with Methylene Chlor ide Organic Layer Concentrate Mu l t i p l e Ex t rac t ions with methylene ch l o r i de Organic l ayer Aqueous l aye r 0 Freeze drying of ur ine samples © Acid hydro lys i s with 1 ml IN hydroch lor ic a c i d , 100°C fo r 1 hour in sealed ampoules in Residue d isso lved methanol and f i l t e r e d Concentrate Methylat ion with methyl -8 —1 reagent Acy la t i on with hepta f luorobutyr i c anhydride 1 © Freeze drying To pH 9.0 with ammonium carbon-ate and ammonium hydroxide Mu l t i p l e ex t rac t ions with methylene ch lo r i de OrganTc Aqueous layer layer a) adjust to pH 12.5 with sodium hydro-xide and hold f o r 1-1.5 hrs . b) br ing down the pH to 9.0 with d i l u t e hydrochlor ide ac id Discard - 75 -were c o l l e c t e d over a per iod of - 32 hours and kept f rozen u n t i l f u r t h e r ana l y s i s. B a s i c a l l y , the experiment cons i s t ed of m u l t i p l e e x t r a c t i o n s (5-6 t imes) of the u r ine sample (15-20 m l ) , us ing approx imate ly 20 ml of methylene c h l o r i d e (a) w i thout any pH adjustments (Step 3) or (b) a f t e r a d j u s t i n g the pH va lue to 9.0 e i t h e r w i th a d i l u t e s o l u t i o n of sodium hydroxide (Step 1) or w i th ammonium carbonate and ammonium hydroxide (Step 2) (ur ine samples were t rea ted w i th a s o l u t i o n ammonium carbonate to r a i s e the pH c l o se to 9.0 (approx imate ly 8.4) and f i n a l pH adjustments were made us ing a d i l u t e s o l u t i o n of ammonium hydrox ide ) . E x t r a c t i o n s were c a r r i e d out i n a sepa-r a t i n g funnel (125 m l ) . The combined methylene c h l o r i d e l a ye r was evapor-ated to dryness under a slow stream of n i t r ogen . Step 6, c ons i s t ed of the procedure descr ibed in Step 2 c a r r i e d out on a c i d hydro lyzed u r ine samples. In Step 4, 15-20 ml of u r ine sample was f r eeze d r i e d (over a per iod of 12 hours) and the res idue d i s s o l v ed in 10-15 ml of methanol. The methanol ic s o l u t i o n was f i l t e r e d and the f i l t r a t e concentrated to about 200 u l . Step 5 c ons i s t ed of the procedure fo l l owed i n Step 4 c a r r i e d out on ac i d hydro lyzed u r i ne samples. The concentrated methanol ic ex t rac t /methy lene c h l o r i d e e x t r a c t i s analyzed using G.L .C . -M.S. system under the f o l l ow i n g c o n d i t i o n s . 2. G.C.-M.S. Cond i t i ons Instrument: MAT 111 equipped w i th a Var ian data base. Column : 1.8 m x 2 mm i . d . c o i l e d g l a s s column packed wi th s i l a r 10 C coated on gas chrom Q (H.P.) 100-200 mesh. MAT 111 G.L .C.-M.S. w i th Var ian data system. - 76 -Operat ing Cond i t i ons : Oven temperature - 100° ( i n i t i a l ) I n j e c t i o n por t temperature - 170° Separator temperature - 280° . C a r r i e r gas (Helium) f low r a t e - 30 ml/min. Temperature Program: I n i t i a l temeprature - 100° Time a t i n i t i a l temperature - 0 min. Rate - 10°/min. F i na l temperature - 250°C and hold u n t i l end of run E l e c t r on vo l tage - 70 eV A c c e l e r a t i n g vo l tage - 820 v o l t s _5 Vacuum - 1 x 10 t o r r 3. De r i v a t i z a t i on a) Me thy la t i on i ) Diazomethane - Diazomethane was generated using e the r , c e l l o s o l v e , Diazald® and potassium hydroxide i n a s p e c i a l l y designed apparatus . The: d i a -zomethane was passed in to the concentrated e x t r a c t u n t i l the s o l u t i o n turned ye l l ow in c o l o r . i i ) Methelute - The concentrated res idue was d i s s o l v ed i n methanol and 100 u l o f methelute was added. The mixture was incubated a t 100°C f o r 6-15 minutes. i i i ) Methyl-8 - To the concentrated res idue in a c en t r i f u ge tube 200 y l o f a c e t o n i t r i l e and 100 y l methyl-8 reagent were added and the s o l u t i o n incubated a t 60° f o r 10 minutes. - 77 -b) A c y l a t i o n The concentrated res idue was d i spersed i n hexane and 200 u l HFBA was added. The s o l u t i o n was kept a t 55° f o r 60 minutes. A f t e r c o o l i n g , i t was evaporated under n i t rogen to remove excess HFBA and r e c on s t i t u t e d i n methanol. F i ve y l o f the d e r i v a t i z e d s o l u t i o n s were i n j e c t e d i n t o a G.C.-M.S. and the f ragmentat ion pat te rns of the peaks ana lyzed . I I . N. TREATMENT OF DATA Plasma Level S tud ies The plasma l e v e l s of t o c a i n i d e i n i n d i v i d u a l r a t s were p l o t t ed aga ins t time and the e l i m i n a t i o n h a l f - l i f e was c a l c u l a t ed from the termina l phase. The area under the plasma l eve l y_s time data (AUC) was c a l c u l a t e d 50 us ing a computer program. The plasma l e v e l data of the i n d i v i d u a l animal 51 52 was ana lyzed by Non-l l in-74 and Autoan. The data were, f i t t e d to-a two compartment open model w i th e l i m i n a t i o n occu r r i ng from the c en t r a l compart-ment. S ince t o ca i n i de e x h i b i t s two compartmental c h a r a c t e r i s t i c s and i t s e l i m i n a t i o n is. n on - l i n e a r , f u r t h e r a n a l y s i s of the d i f f e r e n t r a te constants of the model (us ing the computer programs), was not c a r r i e d out due to the l i m i t e d amount of data from each an ima l . 5 0 H.P.-65 "AUC" tape. 5 1 Non l i n . by Me tz l e r C M . , May 1974. Autoan Manual, by Sedman, A . J . and Wagner, J . G . , May 1976. - 78 -Ur ina ry Exc re t i on Stud ies The percent of the dose excreted as i n t a c t drug i n the 0-24 hour ur ine sample was c a l c u l a t e d from the amount of i n t a c t drug excreted i n the ur ine in t h i s t ime per iod/dose admin i s te red . Both the plasma and u r i ne l e v e l data were analysed f o r the ex i s t ence o f non - l i nea r k i n e t i c s . The s tuden t ' s " t " t e s t was used to t e s t the s t a t i s t i c a l s i g n i f i c a n c e of the d i f f e r e n c e of va r ious treatments a t a = 0 .01. - 79 -I I I . RESULTS AND DISCUSSION A. CONFIRMATION OF THE PURITY AND THE IDENTITY OF MATERIALS 1. P u r i t y and I d e n t i t y of the Chemicals Mass spec t roscop i c a n a l y s i s was used as a means of i d e n t i f y i n g a l l the compounds used in the present i n v e s t i g a t i o n . Compounds were i d e n t i f i e d by t h e i r mo lecu lar ion and the r e spe c t i v e f ragmentat ion p a t t e r n . The i n t e n -s i t i e s of the mass ions observed are tabu la ted in Appendix A. The p u r i t y of a l l of the compounds used was determined by the meas-urement of the me l t i ng po in t and comparing i t w i th the repor ted l i t e r a t u r e va l ues . Furthermore, i n the case of t o ca i n i de hyd roch l o r i de , a s i n g l e endothermic peak corresponding to the t o ca i n i d e hydroch lo r i de was obta ined using the D.S.C. ( F i g . 8). / Organic phase to 10 ml tube > s Evaporate to d ry -ness / 20 y l methylene c h l o r i d e + 2 y l HFBI VORTEX In j e c t 2 1 i n t o GLC Methylene Ch lo r i de 5 ml Column: 1.8m x 2 mm 3% OV 17 on 80/100 mesh chromosorb W cond i t i oned w i th S i l y l - 8 ( P i e r ce ) Temperature: Oven - 250°C I n j e c t o r - 275°C Detector - 275°C Gas f l ow: C a r r i e r gas (Helium) - 30 ml/min. A i r - 300 ml/min. Hydrogen - 30 ml/min. 'Sc heme 4 Mod i f i ed F . I .D . -G .L .C . Ana l y s i s o f Toca in ide Blood In te rna l Standard 0.1 ml Bu f f e r pH 10.0 3 ml Water q . s . 5 ml Cent r i f uge Plasma 1-2 ml Cent r i fuge tube Methylene Ch lo r i de 5 ml Shake f o r 30 min. i n a w r i s t a c t i on shaker Cen t r i f uge to separate l a ye r s 2 ml methylene c h l o r i d e Hexane 1 ml ^Evapora te « HFBA 10 y l > React ion a t 55°C f o r 20 min. < I n j e c t 1-3 nl i n G.C. Column - 1.8m x 2 mm 3% 0V 17 on 80/100 mesh chromosorb W (H.P.) Temperature - Oven - 180°C I n j e c t i o n port - 200°C > Detector - 300°C Gas f l ow - c a r r i e r gas (Helium) - 40 ml/min. a i r - 300 ml/min. hydrogen - 40 ml/min. Retent ion t ime: t o ca i n i d e - 5.79 min. i n t e r na l standard - 7.83 min. - 86 -F igure 11: Flame i o n i z a t i o n de tec to r gas chromatogram:. of t o c a i n i d e (a) and W-49167 (b) obta ined from As t r a Pharmaceut ical P roducts , Inc . - 87 -A t y p i c a l chromatogram obta ined from F . I .D . -G . L .C . a n a l y s i s i s shown i n F igure 12. The peak a t 4.18 minutes corresponds to t o ca i n i d e and tha t a t 5.76 minutes corresponds to the i n t e r na l s tandard . The minimum de t e c t -able amount of t o ca i n i de by t h i s method was 3 ng and the standard curve was l i n e a r over the range of 3 ng to 600 ng (Table V I I I ) . Th is method was used to determine the blood l e ve l o f t o c a i n i d e in a p a t i e n t r e c e i v i n g t o ca i n i d e hydroch lo r ide f o r the treatment of v e n t r i c u l a r arrhythmias ( F i g . 13) . 2. E . C D . - G . L . C . Ana l y s i s a) L i m i t a t i o n s of F . I .D . -G . L .C . Ana l y s i s In s p i t e of the improvements of the F . I .D . -G .L .C . a n a l y s i s , r e l a -t i v e l y l a rge volumes of blood samples (1-5 ml) were necessary f o r a n a l y s i s us ing t h i s .technique, due to inherent s e n s i t i v i t y l i m i t a t i o n s of the F . I .D . -G.L.C. a n a l y s i s . The use of r a t as an animal model imposed r e s t r i c t i o n s on the volume of blood sample tha t cou ld be obta ined dur ing r ou t i ne e x p e r i -ments. Th is prompted the search f o r a more s e n s i t i v e a n a l y t i c a l techn ique. E l e c t r on capture de tec to r s are gene ra l l y more s e n s i t i v e than f lame i o n i z a t i o n de t e c t o r s . The chemical nature of t o c a i n i d e does not lend i t s e l f to easy de tec t i on wi th g rea te r s e n s i t i v i t y by e l e c t r on capture de tec t i on methods. Toca in ide i s amenable to the format ion of d e r i v a t i v e s which are e a s i l y de tec tab le in t race q u a n t i t i e s by E.C.D.-G-.L.C. The development o f an E . C D . - G . L . C . assay technique f o r the development of t o ca i n i de i n b i o -l o g i c a l f l u i d s was the re fo re i n i t i a t e d . Informat ion obta ined from the F . I .D . -G .L .C . a n a l y s i s was very usefu l i n t a i l o r i n g the developmental aspects of a new E . C D . - G . L . C . a n a l y s i s - i n p a r t i c u l a r , column s e l e c t i o n and the e x t r a c t i o n procedure to be f o l l owed . - 88 -F igure 12: Flame i o n i z a t i o n detec tor -gas chromatogram o f t o c a i n i d e (a) and W -49167 (b) obta ined by mod i f ied method. The peak a t 4 . 1 8 i s t o c a i n i d e , the peak a t 5 . 7 6 i s i n t e r n a l s tand-ard ( W - 4 9 1 6 7 ) . Column used 3% O V - 1 7 . TOCAINIDE LEVEL IN A PATIENT F igure 13: Plasma l e v e l s of t o ca i n i de (expressed as base) i n a pa t i en t r e c e i v i n g t o ca i n i d e hydroch lor ide o r a l l y - 90 -Table VI I I Es t imat i on of II Added to Plasma by F lame-Ion iza t ion . Detector GLC Ana l y s i s Amount Mean Added, ng Area R a t i o 9 SE_b 2.64 0.05 0.013 5.27 0.10 0.005 10.6 0.22 0.003 15.3 0.31 0.007 21.1 0.44 0.003 52.7 1.19 0.052 105 2.25 0.019 158 3.85 0.098 211 4.96 0.257 527 12.0 0.158 791 15.4 0.082 1054 19.. 5 0.379 aMean o f three va lues; y = mx + c, where m = 0.229 and c = 0.0056. Standard e r r o r - 91 -b) Optimal C o n d i t i o n s . f o r D e r i v a t i z a t i q n A number of f a c t o r s must be taken i n t o c on s i de r a t i on i n c a r r y i n g out the d e r i v a t i z a t i o n r e a c t i o n f o r gas chromatographic a n a l y s i s , v i z . , i ) the chemical c h a r a c t e r i s t i c s of the d e r i v a t i z i n g agent i i ) the amount of the reagent to be used i i i ) the so l ven t f o r the r e a c t i o n i v ) the temperature a t which the r e a c t i o n i s to be c a r r i e d out v) the time o f i ncuba t i on r equ i r e d , a t the se l e c t ed tem-pera tu re , f o r opt imal r e a c t i o n v i ) the s t a b i l i t y of the d e r i v a t i v e formed v i i ) c on f i rma t i on of the s t r u c t u r e of the d e r i v a t i v e formed The preceding f a c t o r s were c a r e f u l l y eva luated to ensure opt imal r e a c t i o n c o n d i t i o n s . i ) S e l e c t i o n of D e r i v a t i z i n g Agent - The choice of the d e r i v a t i z i n g agent depends on the s e n s i t i v i t y of d e t e c t i o n , v o l a t i l i t y of the r e s u l t i n g product and the ease of p r epa ra t i on . The s e n s i t i v i t y of de te c t i on by an e l e c t r on capture method, inc reases in the f o l l ow i ng order [92] F < N0 2 * Cl < Br < I . However, the f l u o r i n a t e d d e r i v a t i v e s are l i k e l y to be more v o l a t i l e and the re fo re can be separated a t low temperature [ 93 ] . In gene ra l , the sen-s i t i v i t y of de te c t i on inc reases as the number o f e l e c t r on cap tu r i ng group-ings i n the molecule i n c r eases . T r i f l u o r o a e e t i c anhydr ide i s most r e a c t i v e and most v o l a t i l e [ 94 ] . Hep ta f l uo robu ty ry l and pen ta f l uo robu ty ry l groups in general confer more v o l a t i l i t y than t r i m e t h y l s i l y l d e r i v a t i v e s . N-penta-f l uo rop rop i ony l and N-hepta f luorobutyry l d e r i v a t i v e s have a l so been repor ted - 92 -to be much more s t ab l e than the corresponding N - t r i m e t h y l s i l y l . d e r i v a t i v e s [95 ] . For most compounds, hepta f1uorobutyry l d e r i v a t i v e s g ive the g rea tes t s e n s i t i v i t y w i th E.C.D. [ 76 ] . In the present s tudy, the hepta f luo ro d e r i v a t i v e was found to be the most s e n s i t i v e and f o r the reasons mentioned e a r l i e r i n the exper imental s e c t i o n , HFBA was used as the agent of cho i ce . i i ) So lven t f o r the React ion - Widely d i f f e r e n t c ond i t i o n s have been used f o r the a c y l a t i o n o f submicrogram q u a n t i t i e s of hydroxy and amino com-pounds f o r gas chromatographic a n a l y s i s . Benzene [97 ] , n-hexane [98 ] , t e t r a -hydrofuron [ 97 ] , e thy l ace ta te [ 99 ] , N, N-dimethylformamnde [100] and acetone [101] have been used as the r e a c t i o n s o l v en t s . The r ap i d and quan-t i t a t i v e a c y l a t i o n of an amine i n benzene s o l u t i o n r equ i r e s the a d d i t i o n o f a c a t a l y s t such as p y r i d i n e [97 ,102] . In the present i n v e s t i g a t i o n methylene c h l o r i d e , benzene, hexane and chloroform>were examined f o r t h e i r s u i t a b i l i t y as r ea c t i on solvents.. In the absence of any c a t a l y s t , hexane y i e l d e d the maximum amount of the d e r i v a t i v e as observed by the h ighes t area r a t i o o f the d e r i v a t i v e / i n t e r n a l s tandard . i i i ) K i n e t i c s of De r i v a t i v e Formation - The temperature of the r e a c t i o n and the time o f i ncuba t i on are two important f a c t o r s c o n t r o l l i n g the r a t e o f fo rmat ion o f the d e r i v a t i v e s . Depending upon the compound to be d e r i v a t i z e d and the so l ven t used f o r the r e a c t i o n , temperatures rang ing from 24° to 150° and times o f i ncuba t i on rang ing from a few minutes to a few hours have been found to be necessary f o r complete d e r i v a t i v e forma-t i o n [90 ,97 ,100 ,103] . In the present s tudy, the k i n e t i c s of d e r i v a t i z a t i o n were s tud ied ati55°and over a time per iod from 20 to 100 minutes. Maximal y i e l d of the d e r i v a t i v e was obta ined i n about 45 minutes. Pro- , longed incuba t i on over 60 minutes, however, was a s soc i a t ed w i th a decrease - 93 -in the y i e l d of the d e r i v a t i v e , p o s s i b l y due to degradat ion of the d e r i v a t i v e . Hence, i t was decided to incubate the samples f o r a per iod of 50 minutes a t 55°C. i v ) Removal o f Excess Reagent - The presence of even t r a ce quan-t i t i e s of the s t r ong l y e l e c t r on cap tu r ing HFBA i n the sample g ives r i s e to severe d i s tu rbances (man i fes t ing as a l a rge so l ven t f r o n t ) i n the chromato-grams and may a l so depress the s tand ing cu r ren t and the ECD response f o r some time [104] . I t was, t h e r e f o r e , e s s en t i a l to complete ly remove the excess reagent and the a c i d formed dur ing the r e a c t i o n . Removal of the excess reagent can be achieved by e i t h e r d ry ing the r e a c t i o n mixture under a gent le stream of n i t rogen or by hydro l yz ing the excess HFBA and n e u t r a l i -z ing the a c i d formed w i th aqueous ammonia [ 90 ] . Use of the l a t t e r technique l ed to a poor y i e l d o f the d e r i v a t i v e , po s s i b l y e i t h e r due to p a r t i t i o n of the d e r i v a t i v e i n to the aqueous l a ye r or t o . t he hyd r o l y s i s of the d e r i v a t i v e . The observa t i on tha t the response due to the d e r i v a t i v e decreased w i th t ime, seems to suggest po s s i b l e hyd ro l y s i s of the d e r i v a t i v e by water . Removal of the excess agent by evaporat ion gave r ep roduc i b l e r e s u l t s and a time pe r i od o f 25-30 minutes was a l lowed f o r complete evaporat ion of the excess reagent . v) S t a b i l i t y of the D e r i v a t i v e - I n i t i t a l s tud ies showed the d e r i v a t i v e formed to be s t ab l e over a per iod of a t l e a s t 4 hours ( F i g . 6 ) . Experiments c a r r i e d out l a t e r i n d i c a t ed the d e r i v a t i v e to be s t ab l e even over a per iod of 4 days. v i ) S t r u c tu re of the De r i v a t i v e Formed - Toca in ide can r eac t w i th HFBA to form a mono-or d i - h ep t a f l u o r obu t y r y l d e r i v a t i v e . An aqueous s o l u -t i o n o f t o c a i n i d e hydroch lo r ide was ex t rac ted and d e r i v a t i z e d as mentioned e a r l i e r . The d e r i v a t i v e formed was analyzed by G.L.C.-mass spectrometry: - 94 -The molecu lar ion observed a t m/e 388, corresponded to a monoheptaf luoro-bu ty ry l d e r i v a t i v e of t o c a i n i d e . Plasma samples from r a t s admin is tered t o c a i n i d e (20 mg/kg) were poo led, ex t rac ted and analyzed by G.L.C.-mass spectrometry f o l l o w i n g d e r i v a t i z a t i o n ( F i g . 14) . The molecu lar ion was observed a t m/e 388 and the mass spectrum w a s ' i d e n t i c a l " t o the one obta ined .a f ter d e r i v a t i z a t i o n of pure t o c a i n i d e . As shown i n Scheme 5, HFBA reac t s w i th t o ca i n i d e to form a monohepta-f l uorobutyry l d e r i v a t i v e and hep ta f l uo robu t y r i c a c i d . The pos tu la ted f ragmentat ion pa t te rn i s shown i n Scheme 6 and was c ons i s t en t w i th the format ion of a monoheptaf luorobutyry l d e r i v a t i v e of t o c a i n i d e . Apar t from the molecu lar i o n , o ther abundant ions were observed at m/e 240, 192, 176, 169, 147, 121 , 119, 105 and 69. • c) Op t im i za t i on of G~L.C. Cond i t i ons i ) E f f e c t of D i f f e r e n t S t a t i o na r y Phases - The two major c r i t e r i a used in s e l e c t i n g the proper column f o r gas chromatographic a n a l y s i s were (1) opt imal r e s o l u t i o n and (2) the shape of the peaks. Table IX and F igure 15 summarizes the responses of the d e r i v a t i v e s of t o c a i n i d e and the i n t e r n a l standard i n d i f f e r e n t columns. With the same extent of l oad ing of the l i q u i d phase as the p o l a r i t y of the column was increased the r e t e n t i o n time of the t o ca i n i de d e r i v a t i v e a l so i n c reased . With OV-225, the t o ca i n i d e d e r i v a t i v e d id not chromatograph wei1 a t a l 1 . Add i t i o n of a smal l amount of OV-225 to 0V-T7 phase tended to inc rease the r e t en t i o n time of the t o ca i n i de peak. Based on opt imal separa t i on of - 95 -#67 —71 RT = 5.37 100 II, , I I M N , 1 1 ' ' I I I I i T"T"t' i f"i |T"i c i"i I , M I I M I " " I i i i 'i r i i i'i i i i i i i ) i i" i | i i i 20 50 100 150 200 250 100 > 250 x 8 10 J i fCH3 p ' C H 3 CH3 O // VNH—C—CH—NH—C—CF2—CF2—CF3 M.W. 388 10 1 1 1 ' 1 i i i i i i i i i i i i i i i i i i "l i i i i i i i i i i i i i i i i i i i i i i 270 300 350 400 520 550 600 F igure 14: Mass spectrum o f t o ca i n i de d e r i v a t i v e obta ined by G.L.C.-mass••s.pect'mffi'etry*:'after e x t r a c t i o n of plasma from r a t dosed w i th t o ca i n i d e (20 mg/kg) Scheme 5 D e r e a l i z a t i o n of Tocainide with Heptafl uorobutyric Anhydride 0 iCH, / — Q ^ - C F j - C F a - C F , ^ N H - C - C H - N H S + ^ N O L I • X H , ' 0 2-AMINO 2 ' -6 ' - PROPIONOXYLIDIDE HEPTA FLUORO BUTYRIC ? A M • • • ANHYDRIDE / C H 3 Q 0- NH-S- r H-<_ c F 8_ c F a_ C F > + C F J . C F 8 . C F 8 J_OH C H , 5 0 H F P T A FLUORO BUTVUYi DERIVATIVE OF H f PTA FLUORO BUTYRIC 2 ' A M I N 0 - 2 ' - 6 ' - P R 0 P I 0 N 0 X Y L I U ! J j b S^iii - 97 -Scheme 6 Mass Fragmentat ion Pa t t e rn o f Toca in ide De r i v a t i v e C H 3 \ \ _NH—C—CH—NH—C—CFo—CFo—CF«J I C H 3 -CH—NH—C—CF 2 —CF 2 —CF 3 C H 3 m/e 388 O ll -CF2—CF2—C—NH—CH—CH3 m/e 240 C H 3 ri * _ 1 ^ y - N H — C — C H — N H 2 1 C H 3 C H 3 m/e 192 C F 3 - C F 2 - C F 2 m/e 169 (/ \—NH—C*—CH 1 C H 3 m/e 176 + C F 3 - C F 2 m/e 119 C H 3 m/e 147 + C F 3 m/e 69 C H 3 C H 3 m/e 105 0~nh C H 3 m/e 120 - 98 -Table I X Response of Toca in ide De r i v a t i v e and In te rna l Standard (g-Bromonaphthlene-)' i n - D i f f e r e n t - S t a t i o n a r y Phases No. Column 1, 3% OV-17 Coat ing Support Chromosorb W (H.P.) 100-120 m Retent ion Time (min.) Toca in ide I n t . S t d . De r i v a t i v e 1.96 4.20 Comments Good r e s o l u t i o n sharp peaks 3% OV-17 (10") 3% OV-225 Chromosorb W (H.P.) 80-100 m 1.77 5.94 Good r e s o l u t i o n but broad de r i va -t i v e peak 3% 0V-25 Chromosorb W (H.P.) 2.66 5.75 Good r e s o l u t i o n but broad de r i va -t i v e peak 3% OV-225 Chromosorb W (H.P.) 80-100 m 1.79 Toca in ide de r i va -t i v e does not chromatograph we l l 10%.0V-101 Chromosorb W (H.P.) 2.5 80-100 m 6.77 Broad d e r i v a t i v e peak 5% 0V-7 Gas.Chrom Q (H.P.) 2.13 80-100 m 6.15 Broad d e r i v a t i v e peak. T a i l i n g a l s o observed GP 3% SP 2250 Supelcoport 100-120 m 1.13 3.51 Broad t o c a i n i d e peak. In te rna l standard peak too c l o se to the s o l -vent f r o n t F igure 15: E l e c t r on capture de tec to r gas chromatograms of a-bromonaphthalene (a) and t o ca i n i de d e r i v a t i v e (b) i n d i f f e r e n t columns under the f o l l ow i ng c ond i t i o n s : oven temperature - 170°; detec tor tem-perature - 300°; i n j e c t i o n port temperature - 200°; c a r r i e r gas f low r a t e - 40 ml/minute A. 3% OV-17; B. 10% OV-17; C. 5% 0V-101; D. 5% SP 2250 - 100 -peaks and symmetrical chromatographic peaks along wi th long column l i f e , OV-17 was se l e c ted as the column o f cho i ce . i i ) E f f e c t o f Oven Temperature and C a r r i e r Gas Flow - The r e s o l u t i o n and the shape o f the peak can be a f f e c t ed by the temperature of the oven and the c a r r i e r gas f l ow r a t e . These two f a c t o r s were manipulated so as to ob ta in sharp peaks w i th maximal r e s o l u t i o n , not on ly of the peaks of i n t e r -es t but a l s o ahy^other peak due to endogenous-mater ia l , metabo l i t e*or impu r i t i e s from so l v en t . i i i ) E f f e c t o f I n j e c t i o n Po r t Temperature - The response and peak shape a re , i n p a r t , dependent upon the temperature a t which the sample i s v o l a t i l i z e d . Too:low an i n j e c t i o n por t temperature causes the sample to be int roduced as a broad band, l ead ing to f u r t h e r peak broadening. Incomplete v apo r i z a t i o n o f the sample may cause condensat ion of the sample on the wa l l of the i n j e c t i o n po r t . On the other hand, too high a temperature may decompose the sample and, hence, decrease the de tec to r response to the compound. S ince by convent ion , the temperature of the i n j e c t i o n por t i s h igher than the oven, the response to the d e r i v a t i v e was monitored over an i n j e c -t i o n por t temperature range o f 200°-300°. Table X shows the response ob ta ined . E s s e n t i a l l y no d i f f e r e n c e i n the response was observed over t h i s range of temperature. A temperature of 200° was chosen as the i n j e c t i o n por t temperature to min imize the r i s k o f decomposit ion and septum b l eed . i v ) E f f e c t of Detector Temperature - The number of e l e c t r ons emit ted from the r a d i o - a c t i v e source, t h e i r energy and the e l e c t r on capture mechanism are i n f l uenced by the de tec to r temperature [104,105] . The re--sponse to the d e r i v a t i v e was s tud ied over a temperature range from 250° to - 101 -• Table X E f f e c t . o f I n j e c t i o n Por t Temperature on the Response o f the De r i v a t i v e Temperature Response 200° 0.99 210° 1.01 220° 1.01 230° 1.02 240° 1.01. 250° 1.03 260° 0.99 270° 1.01 280° 1.00 290° 1.00 300° 0.98 a Area r a t i o o f t o c a i n i d e d e r i v a t i v e / i n t e r n a l s tandard n = (2-3) determinat ions at each temperature. - 102 -350°. This range was chosen s i n ce above 350° the l i f e o f the de tec to r i s shortened and below 250° the de te c to r becomes contaminated e a s i l y . Table XI shows the responses obta ined at d i f f e r e n t temperatures. At temperatures lower than 280°, the response decreased d r a s t i c a l l y and a t 250°C the response was on ly 81% o f t ha t at 300°C. S ince i n e a r l i e r s t u d i e s , the r a t i o decreased s l i g h t l y at 350°C, 300°C was taken as the optimum temperature. d) Op t im i za t i on of E x t r a c t i o n i ) So l ven t E f f i c i e n c y - The three f a c t o r s taken i n t o c ons i de r a t i on i n dec id ing the so l ven t of cho ice f o r e x t r a c t i o n are as f o l l ow s : (1) E x t r a c t i o n e f f i c i e n c y (2) V o l a t i l i t y , and (3) P u r i t y F igure 16 shows the e x t r a c t i o n e f f i c i e n c y of d i f f e r e n t so l ven ts t e s t ed i n t h i s s tudy. The order of e f f i c i e n c y was as f o l l o w s : e ther > methylene c h l o r i d e > ch loro form > ethy lene c h l o r i d e > hexane > benzene. Ether was too v o l a t i l e to handle e f f e c t i v e l y , and methylene c h l o r i d e was v o l a t i l e enough to be evaporated f a s t e r than o ther s o l v en t s . Moreover, s i nce methylene c h l o r i d e was obta ined i n a h i gh l y pure form, i t was s e l e c t ed as the so l ven t o f cho i ce . i i ) Recovery S tud ies - Table XII shows the standard curve obta ined in E .C.D.-G.L .C. The recovery data areshown i n Table X I I I . Recover ies were c a l c u l a t e d by comparing the responses of the base which was d e r i v a t i z e d w i thou t any e x t r a c t i o n w i th the ex t r a c t ed s a l t s o l u t i o n . Use o f the t o ca i n i d e base obta ined by e x t r a c t i o n w i th benzene y i e l d e d a recovery value of 67.08% ± 5.43. When the base obta ined us ing methylene c h l o r i d e e x t r a c t i o n was used, h igher recovery va lues were obta ined (78.00% ± 9 .50) . - 103 -Table XI E f f e c t o f Detec tor Temperature on the Response of the De r i v a t i v e Temperature 250 260 270 280 300 310 320 330 340 350 Response 1 0.45 0.47 0.50 0.52 0.55 0.57 0.59 0.59 0.59 0.58 n = 3 at each temperature a Area r a t i o of t o c a i n i d e d e r i v a t i v e / i n t e r n a l s tandard [4] i o o o o o o o o o o o o o o o o o [6] [4] [3] " f c l A A [3] 0 o A + o o A a V y 0 ° o 0 A A A V A V o 1A A V A ether ethylene met ehloride chloride lene henane chloroform benzene F igure 16: Ex t rac t i on e f f i c i e n c y of d i f f e r e n t so lvents . A l l values expressed as mean ± standard dev i a t i on . The number In the parentheses re fe rs to the number of determinations ca r r i ed out - 105 -Table XII Es t imat i on o f Toca in ide by E l e c t r on Capture Detector Gas Chromatography (standard cu rve ) Amount Added, ng n_ Mean Area R a t i o 9 0.18 1 0.034 -0.35 6 0.072 . 0.002 0.70 5 0 . 1 6 1 " 0.002 1.05 ; 6 0.263. 0.005 1.40, 6 0.33. . 0.002 1 .75 3 0.373 0.002 2.10 2 0.490 0.009 2.45 6 .0.694 . 0.003 2.80 4 0.716 . :• 0.002 a With y = mx + c, where m = 0.264 and c = -0 .027. Where m = s lope and c i s the i n t e r c ep t k Standard e r r o r - 106 -Table XI I I Recovery of II i n Rat Plasma as Determined by the E lec t ron-Capture Detector GLC Ana l y s i s Amount o f II Added to 0.1 ml o f Plasma, ng ii 0,18 1 0.35 6 0.70 5 1.05 6 1.40 6 1.75 3 2.10 2 2.45 6 2.80 4 Mean recovery Amount of II Recovered a f t e r E x t r a c t i o n , ng 0.13 0.22 0.46 0.73 0.91 1.02 1.33 1.88 1.93 Mean Recovery, % 72 64 66 70 65 58 63 77 69 67 SD o f Percent Recovery 10.6 5.0 8.0 2.4 0.7 2.0 1.9 0.7 5.0 - 107 -The d i f f e r en ce s i n these va lue can be a t t r i b u t e d to the f o l l o w i n g reasons: (1) the base obta ined a f t e r methylene c h l o r i d e showed the presence of an ad-d i t i o n a l peak i n the d i f f e r e n t i a l scanning ca lor imeter" . Th is suggested the presence of a po s s i b l e impur i t y or incomplete evaporat ion of the s o l v e n t , r e s u l t i n g in a r e l a t i v e l y h igher recovery when t h i s base used f o r recovery s t ud i e s ; (2) the base obta ined a f te r , methylene c h l o r i d e seemed to have a s l i g h t l y lower s o l u b i l i t y than the one obta ined a f t e r benzene e x t r a c t i o n . e) G.L.C. of Plasma and Ur ine Samples Representa t ive chromatograms from the e x t r a c t s of plasma and u r ine samples are shown i n F igure 17. The peak a t 2.09 corresponds to the i n t e r na l standard and the one a t 4.53 to the t o ca i n i de d e r i v a t i v e . The other peaks were e i t h e r due to endogenous substances present i n the plasma or to the impu r i t i e s i n the so l ven t used f o r the a n a l y s i s . These extraneous peaks, however, d i d not inter- fere w i th the q u a n t i t a t i o n o f the compound of i n t e r e s t . The minimum de tec tab le amount by t h i s method was about 30 peg. The response was observed to be l i n e a r over a range of 50 p eg / i n j e c t i o n to 3 n g / i n j e c t i o n (0.39 to 4.0 yg/ml p lasma). f ) Ana l y s i s o f Faecal Samples The chromatogram obta ined from the E .C.D.-G.L .C. a n a l y s i s of the blank faeces i s shown in F igure 17. A small peak was observed a t a r e t en -t i o n time (4.73 min.) which was very c l o se to the r e t e n t i o n time of the peak due to the t o ca i n i de d e r i v a t i v e (4.12 m i n . ) . The blank faeces sample however d i d not show any peak in F . I .D . -G . L .C . a n a l y s i s . The faeca l samples c o l l e c t e d a f t e r in t ravenous a dm i n i s t r a t i o n o f t o c a i n i d e to r a t s were analyzed by both F . I .D . -G . L .C . and E .C .D. -G.L .C . The minimum amount o f - 108 -a » 9 I L L b 2 b y Figure 17: Electron capture detector-gas chromatogramfoof a-bromonaphthalene (a) and tocainide der ivat ive (b) in 3% OV-17 A - Blank plasma C - Blank urine E - Blank faeces B - Plasma extract containing tocainide D - Urine extract containing tocainide F - Aqueous extract containing tocainide - 109 -t o ca i n i d e tha t cou ld be detected us ing the F . I .D . -G .L .C . a n a l y s i s method adopted i s approx imate ly 0.42-0.85% of the dose adm in i s t e red . The E . C D . - G . L . C . method adopted in the present study cou ld de tec t up to 0.014-0.028% of the dose admin i s t e red . g) V a r i a t i o n s i n the A n a l y t i c a l Methodology Use of IN, 2N or 5N sodium hydroxide d id not a f f e c t the recovery of t o c a i n i d e . I t was a l so observed tha t a d d i t i o n of up to 0.5 gm of NaCl to 1 ml blood or u r ine sample before e x t r a c t i o n a l s o had no e f f e c t on the recovery.^ The plasma to whole blood p a r t i t i o n c o e f f i c i e n t was found to be 1.20 ± 0 .08 - J • Because of the d i f f i c u l t i e s a s soc i a t ed w i th e x t r a c t i o n of b lood i t was p re fe r red to ana lyze the plasma samples as the p a r t i t i o n c o e f f i c i e n t was c l o se to 1.0. h) In te r f e rence by Other Compounds Ne i the r normal components of the blood or u r ine nor the f o l l ow i n g drugs i n t e r f e r r e d w i th the assay procedure: metoclopramide, SKF 525-A, s a l i c y l a m i d e , g r i s e o f u l v i n , d i a z epam, g l y c i n e x y l i d i d e and l i d o c a i n e ( l i d o -ca ine cannot be d e r i v a t i z e d and i s , hence, not detected by e l e c t r on capture de tec to r i n small q uan t i t i e s ) . ( F i g . 18) . i ) Comparisons of F.I .D . -G .L .C . vs E . C D . - G . L . C . Table XIV summarizes some parameters of i n t e r e s t of the F . I .D . -G . L .C . and the E . C D . - G . L . C . a n a l y s i s o f t o c a i n i d e . The minimum de tec tab l e l e v e l of t o ca i n i de by E . C D . - G . L . C was 60 times lower than tha t us ing F . I .D . -G . L .C . From As t r a Pharmaceut ical Co. I n c . , Framingham, Massachuset ts . - n o -F igure 18: E l e c t r on capture de tec to r -gas chromatograms of some drugs to t e s t f o r po s s i b l e i n t e r f e r en ce in the a n a l y s i s A. G r i s e o f u l v i n : (oven temperature 250°) 1.6 ng B. MetQclopramide (oven temperature 235°) 1.0 ng C. Sa l i c y l am ide (oven temperature 180°) D. SKF 525-A (oven temperature 180°) E. Diazepam (oven temperature 180° f o r 5 m ins . , r a t e 80°/min. to 260° and hold) F. G l y c i ne x y l i d i d e (oven temperature 180°) - i n -Table XIV Comparison of E .C .D.-G.L .C. vs F . I .D . -G . L .C . F . I .D . -G . L .C . E .C .D. -G.L .C . Minimum de tec tab l e l e ve l 3 ng 50 peg L inea r Range 3-1000 ng 100.peg-3 ng Plasma volume r e -qu i red f o r a n a l y s i s 0.5-1.0 ml 50-100 y l - 112 -method. The standard curve was l i n e a r from 100 peg to 3 ng in the E.C.D.-G.L.C. :,analysis and from 3 ng to 1000 ng in the F.I .D . -G .L .C . a n a l y s i s . A 10 f o l d l a r g e r plasma sample was requ i red f o r the F . I .D . -G .L .C . a n a l y s i s due to s e n s i t i v i t y l i m i t a t i o n s . I I I . C. SELECTION OF ANIMAL MODEL Ease o f hand l i ng , c o s t , s i z e and the s u i t a b i l i t y to the i n v e s t i g a t i o n under c on s i de r a t i on o f ten d i c t a t e s the s e l e c t i o n of a p a r t i c u l a r animal model f o r pharmacokinet ic and metabo l i c s t u d i e s . Monkeys, dogs, r a b b i t s , guinea p i g s , mice and r a t s are some of the commonly used animals i n drug metabol ism s t ud i e s . The lack of f a c i l i t i e s prec luded the use of monkeys and dogs f o r the present i n v e s t i g a t i o n . Moreover, dogs have been shown to lack a c e t y l a t i n g enzymes [106] . Rabbi ts have been shown to be a poor cho ice in abso rp t i on s tud ies due to the sub s t an t i a l d i f f e r en c e in the g a s t r o i n t e s -t i n a l phys io logy [107] . S e r i a l samples, f o r determin ing the k i n e t i c s of d i s p o s i t i o n of a drug, cannot be r e a d i l y obta ined us ing mice. " I t i s r e l a -t i v e l y d i f f i c u l t to produce enzyme i nduc t i on i n the mouse, wh i l e the r a t i s a more respons ive an ima l . A l though, on ly a few compounds have been e va l u -ated i n man, i t would appear tha t man more c l o s e l y resembles the r a t i n t h i s regard" [108] . Hence, the r a t was chosen as the model system f o r the present i n v e s t i g a t i o n . Procedure f o r Obta in ing Blood Samples T a i l Ve in : Small volume> biood-.samples can.be obta ined from the t a i l ve in of the r a t . However, f o r c o l l e c t i o n of samples, the r a t s have to be e i t h e r - 113 -r e s t r a i n ed manual ly or anaes the t i s ed . A number of b iochemical parameters such as blood a c i d i t y , plasma p r o t e i n s , M g + + , and C a + + ions and blood g lucose l e v e l s have been shown to be a l t e r e d when manual r e s t r a i n t i s used to c o l l e c t the blood samples [109] . Of the var ious methods of induc ing anaes thes i a , the use of e ther i s the most common • one, because of i t s ease of a d m i n i s t r a t i o n . With e ther as an anaes t he t i c , the animal undergoes a phase of a g i t a t i o n before going i n t o anaes thes i a . Ether a l so s t imu l a t e s s e c r e t i on i n the an ima l s . I t tends to remain i n body f a t f o r a per iod of t ime a f t e r anaesthes ia arid c i r c u l a t o r y adapta t ion occurs a f t e r the use of e ther [110] . In v i t r o , ether d i d not have any s t i m u l a t i n g e f f e c t on the a c t i v i t y of the r a t l i v e r microsomal enzymes [111] , whereas i n v i vo s tud ies have shown ether to be a potent s t imu l a t o r o f the microsomal enzymes [112] . Exposure to a sub anaes thes t i c concen t ra t i on of d i e t h y l e t h e r has been demonstrated to enhance the a b i l i t y of r a t s to metabo l i ze hexobarb i ta l [112] . Moreover, accurate t im ing o f the sampling i s not always po s s i b l e when us ing e the r . There may a l so be l o s s of some blood from the punctured area of the t a i l . For the above mentioned reasons, i t was des i r ed to f o l l ow a method o f blood sampling which does not i nvo l ve the use of any anaesthe-t i s i n g agent. The technique of j u g u l a r ve in cannu la t i on proved very s u i t -ab le f o r the purposes of the s tudy. The" use of a j u gu l a r ve in cannula f a c i l i t a t e s a dm in i s t r a t i o n of drug s o l u t i o n as .we l l as withdrawal of blood samples a t des i red t i m e . i n t e r v a l s . In a l l the s tud ies , Wis tar r a t s weighing about 190-210 gms were used. The blood volume in these r a t s :has been shown to be around 9-10 ml [113] . - 114 -During the exper imenta l pe r iod of 12 hours near l y 2.5 ml o f the blood volume i s withdrawn from the an ima l . Th is i s to avo id the p o s s i b i l i t y of any a l t e r -t i o n in the haemodynamic parameters. For the same reason, separate r a t s were used f o r each study r a the r than on a cross over b a s i s . I I I . D. PHARMACOKINETICS OF TOCAINIDE 1. Plasma Level S tud ies Determinat ion of the time course of the plasma l e v e l s of a compound a f t e r in t ravenous adm in i s t r a t i o n prov ides sub s t an t i a l i n fo rmat ion about the d i s t r i b u t i o n and e l i m i n a t i o n c h a r a c t e r i s t i c s of a compound. In order to f u l l y understand the k i n e t i c s of the drug, i t i s necessary to c a r r y out s i n g l e dose int ravenous s tud ies over a wide range of dose l e v e l s . Such s tud ies w i l l prov ide the necessary i n fo rmat i on to determine the l i n e a r i t y or the n o n - l i n e a r i t y of the d i s t r i b u t i o n or the e l i m i n a t i o n processes . In the present i n v e s t i g a t i o n , plasma concen t ra t i on time s tud ies were c a r r i e d out i n r a t s f o l l ow i n g in t ravenous adm in i s t r a t i o n of 5, 10, 15, 20 and 40 : ;mg/kg of t o ca i n i de hydroch lo r i de (expressed as base) . F igure 19 shows the semi logar i thmic p l o t of the plasma concen t ra t i on _vs time (PCT) data f o l l ow i ng 10 mg/kg dose. Table XV summarizes the PCT data i n a number of a n ima l s . f o l l ow i ng 10 mg/kg dose of t o c a i n i d e . The k i n e t i c s of t o c a i n i d e cou ld be desc r ibed by a two compartment open model w i th a d i s t i n c t but shor t d i s t r i b u t i o n phase fo l l owed by an e l i m i n a t i o n phase. The d i s t r i b u t i o n h a l f - l i f e was 13 minutes and the . e l i m i n a t i o n h a l f - l i f e was about 83 minutes. » 120 2 4 0 3 6 0 4 8 0 T I M E (min) Figure 19: Plot of plasma concentration (mean t standard deviation) vs tine, following Intravenous administration of tocainide (10 mg/kg) to 4 rats - . 1 1 6 -Table XV Plasma Level vs Time Fo l l ow ing Intravenous Adm in i s t r a t i o n of 10 mg/kg of Toca in ide Time : Rat #1 Rat #2 ( '," Rat #3 Rat .#4 Hours 0 .17 yg/ml 2 . 1 7 yg/ml , 2 . 6 3 yg/ml 3 . 2 3 yg/ml 2 . 5 0 0 . 3 3 2 . 0 0 . 2 . 5 9 . ' 2 . 9 9 2 . 0 9 o . 5 - - 2 . 6 8 / 1 . 0 5 / 1 . 0 , 1.17 • 1,82 , 2 . 0 0 "'• 0 . 9 0 1 . 5 , 0 . 9 9 0 . 9 3 - -2.0.' - 0 . 6 7 - 1.23 0 . 7 8 2 . 5 0 . 9 0 - 0.51 3 . 0 • - 0 . 6 5 • 1.05 • -3 . S •"' 0.51 - - -4 . 0 . 0 .24 : - 0 . 5 5 0 . 4 5 6 . 0 . 0 . 2 5 o.oa • .0.44 -8 . 0 ' , 0 . 1 7 0.03 , 0 . 1 3 0 . 1 2 - 117 -F igure 20 shows the semi l oga r i t hm i c p l o t of the plasma concen t ra t i on vs time data f o l l o w i n g in t ravenous adm in i s t r a t i o n of 20 mg/kg (expressed as base) of t o c a i n i d e hyd roch l o r i de . Table XVI summarizes the plasma l eve l data on f i v e r a t s f o l l ow i ng int ravenous adm in i s t r a t i o n of 20 mg/kg o f t o c a i n i d e . The k i n e t i c s of t o ca i n i de a t t h i s dose l e ve l cou ld again be descr ibed by a two compartment model. The d i s t r i b u t i o n h a l f - l i f e was 10 minutes and the e l i m i n a t i o n h a l f - l i f e was 156. minutes. F igure 21 shows the semi logar i thmic p l o t of plasma concen t ra t i on vs_ time data f o l l ow i ng in t ravenous adm in i s t r a t i o n of 5, 10, 15, 20, and 40 mg/kg of t o ca i n i de in f i v e d i f f e r e n t an ima l s . Up to a dose l e ve l of 15 mg/kg the plasma h a l f - l i f e was about 83 minutes. At and above 20 mg/kg dose l e ve l there i s a tendency f o r the b i o l o g i c a l h a l f - l i f e to i n c r ease . Thus an inc rease in the time requ i red f o r the drug concen t ra t i on to de-c l i n e by 50% of the o r i g i n a l concen t ra t i on was observed a t and above 20 mg/kg dose l e v e l . When the e l i m i n a t i o n process f o l l ows s imple f i r s t order k i n e t i c s , the termina l phase of the plasma concen t ra t i on vs_ time data de s c r i b i ng the e l i m i n a t i o n k i n e t i c s of drug w i l l be p a r a l l e l a t d i f f e r e n t dose l e v e l s . (The b i o l o g i c a l h a l f - l i f e ( t - j^ ) W 1 '^ be independent o f the dose.) Hence, i t appears tha t the e l i m i n a t i o n process of t o ca i n i de i n r a t cannot be de-s c r i b ed by a s imple f i r s t o rder l i n e a r d i f f e r e n t i a l equa t i on . The d i f f e r e n t k i n e t i c parameters of t o c a i n i d e are shown i n Table XVI I . In a d d i t i o n , the area under the plasma l e v e l time curve (AUC) was observed to inc rease in a d i s p r opo r t i o na t e manner w i th the dose ( F i g . 22 ) . When l i n e a r k i n e t i c s o f e l i m i n a t i o n are i n ope r a t i on , the va lues of AUC inc rease w i th the dose i n a p ropor t i ona te manner, accord ing to equat ion (1) f o r a one compartment model and equat ion (2) f o r a two compartment model - 118 -120 240 360 480 TIME (min) F igure 20: Semi logar i thmic p l o t o f plasma concen t ra t i on (mean ± standard dev i a t i on ) vs time f o l l ow i n g i n t r a v enou s ' a dm i n i s t r a t i o n of t o c a i n i d e hydro-c h l o r i d e (20 mg/kg) to r a t s - 119 -Table XVI Plasma Concent ra t ion v$:Time;->following Intravenous Adm in i s t r a t i o n o f 20 mg/kg o f Toca in ide Time Rat #1 Rat #2 Rat #3 Rat #4 Hours ug/ml yg/ml yg/ml yg/ml 0.17 15.0 l ( 9.77 9.26 11.22 . 0.33 . 7.87' 8.57 8.58 6.92 0.50 - 7.29 4 .97 - 9.38 1.0 . 6.86; 5.93 / 4.87". 7.04 1.5 4.26 3.77 . 3.95 . 4.34 2.0 ; 4.. 06 '•• 3.14 3.60 2.5 . - - 3.33 3.52 3.0 v; - - 1.96 -4.0 1.38 ' 2.06 2.16 .- 3.00 6.0 . _ - 1.52 -8.0 . 0.46 ' 0.17 0.83 1.61 -120-..,,.U.U..I.U»I. . , . ,»^Au.u. , . I , „ , i ' n i „ . „ . „ f 120 240 360 4 8 0 T I M E (min) F igu re 21: Semi logar i thmic p l o t o f plasma c on cen t r a t i o n versus t ime , f o l l ow i n g in t ravenous a d m i n i s t r a t i o n of (•) 5 mg/Kg, (•) 10 mg/Kg, (A) 15 mg/Kg and (o) 20 mg/Kg o f t o c a i n i d e . Each curve i s the mean of 3-5 an ima l s . Bars represent s tandard dev i a t i o n s from the mean. - 121 -Table XVII K i n e t i c Parameters Fo l l ow ing Intravenous Adm in i s t r a t i o n o f Toca in ide to Rats Dose (mg/kg) 5 10 15 20 Amount o f drug .D i spos i t i on Admin is tered ra te const . H a l f - L i f e i n 24 hours (mg) (minute-1) (minute) (yg) 2 ' Amount of i n -t a c t drug ex-c re ted i n u r ine Renal . _ C learance 0 (ml/min.) 0.0083 161± IT 83±12u 364± 90 0.0044 156 533± 121 1490± 308 1.20 0.87 0.85 1.07 40 0.;0050 143 4084±1008 1.30 Ca l cu l a t ed from the equat ion Renal c l ea rance i n t a c t drug i n ur ine area under plasma l e ve l time curve . Mean ± standard dev i a t i o n D O S E - A U C RELATIONSHIP A F T E R i.v. ADMINISTRATION (4) 20 DO 0 ' ' 3 O 10 .-•NT I p < (6) (3) W r o r o 0 F igure 22: 10 15 20 DOSE IN mg/kg P l o t of area under the plasma concent ra t ion vs time curve [/ cpdt = AUC] aga ins t dose of t o c a i n i d e admin is tered in t ravenous ly to rat§. The va lues are mean ± standard d e v i a t i o n . The numbers i n parentheses r e f e r to the number of animals a t each dose. - 123 -AUC = D (1) AUC = D (2.) 3Vd area where AUC i s the area under the plasma l e v e l time curve , D i s the dose admin i s te red , K £ i s the e l i m i n a t i o n r a t e constant from a one compartment model, 3 i s the hybr id e l i m i n a t i o n r a t e constant and Vd i s the volume of d i s t r i b u t i o n . The va lue o f D/AUC which i s r e f e r r e d to as the t o t a l body c l ea rance w i l l be constant when l i n e a r k i n e t i c s of e l i m i n a t i o n are i n opera t i on [114] . As can be seen i n : the Table XVIII the c l ea rance va lue ( t o t a l body c learance) decreases w i th an inc rease i n the dose. At 5, 10 and 15 mg/kg dose l e v e l s the c learance i s about 4 .5-5 .0 ml/min. Th is va lue decreased to about 3 ml/ minute a t 20mg/kg dose.Thus the i n c rease i n the AUC f o l l ow i n g 20 mg/kg dose i s l i k e l y to be due to the r e s u l t o f a reduc t i on i n the t o t a l body c learance of t o c a i n i d e . The preceeding observa t ions i nd i ca te " that" a . s i m p l e . l i n e a r pharmaco k i n e t i c model i s inadequate to desc r i be the k i n e t i c s of t o c a i n i d e and tha t a non- l i nea r model would be more app rop r i a t e . Chan [115] developed a general theorem f o r s tudy ing the n o n - l i n e a r i t y of area-dose r e l a t i o n s h i p . Depending upon the nature of the process respon-s i b l e f o r the n o n - l i n e a r i t y (Langmuir type b ind ing o r M ichae l i s -Menten e l im i n a t i o n ) d i f f e r e n t r e l a t i o n s h i p s .were observed between area-dose r e l a t i o n -sh i p . In the case of M ichae l i s -Menten e l i m i n a t i o n a convex area-dose and a r e a - i n i t i a l concen t ra t i on r e l a t i o n s h i p were observed. With Langmuir type b ind ing the a r e a - i n i t i a l concen t ra t i on r e l a t i o n s h i p was concave and the - 124 -Table XVIII Add i t i ona l Pharmacokinet ic Parameters o f Toca in ide i n Rat Dose mg/kg n Amount Admin is tered mg — oo a AUG] Q mg.hr ml~^ Tota l Body C l ea rance 3 (C l ) ml /mi n 1 Volume o f D i s t r i b u t i o n (v B ) ml 5 4 1 2.23±0.72 7.4±1.7 886±206 10 5 2 6.95±2.14 5.1+1.6 615±193 15 3 3 10.43±0.43 4.8±0.6 577±,71 20 6 4 23.13±4.2 2.8±0.5 b' 644±117 40 4 8 56.31±16.6 2.50±0.7b 516±179 ' a Mean ± standard d ev i a t i o n b S i g n i f i c a n t l y d i f f e r e n t from 5, 10, 15 mg/kg dose l e v e l s p < 0.01 - 125 -area-dose r e l a t i o n s h i p was l i n e a r . These theo r i e s were based upon a one compartment model and cannot be d i r e c t l y ex t rapo la ted to m u l t i -•compartment models. The nature of the a r e a - i n i t i a l concen t ra t i on r e l a -t i o n s h i p i s i n f luenced by the d i s t r i b u t i o n a l parameters of the drug as w e l l . 2V Ur inary Exc r e t i on Stud ies a) Spec ies D i f f e rence i n the Ur inary Excret ion ' .o f Toca in ide Table XIX summarizes the data on the percent of dose admin i s te red tha t i s excreted as i n t a c t drug i n the 24 hour time pe r i od , a f t e r o r a l a dm in i s t r a t i o n of t o c a i n i d e , i n d i f f e r e n t spec ies o f an ima l s . In humans, a t dose l e v e l s l e s s than 15 mg/kg approx imate ly 40% of the dose i s excreted as i n t a c t drug i n the 24 hour u r i ne sample. In r a t s , a dose of 15 mg/kg r e su l t e d i n approx imate ly 17% of the dose being excre ted as i n t a c t i n the 24 hour time pe r i o d . However, a t a dose of 20 mg/kg, near l y 32% of dose admin i s te red i s recovered as i n t a c t drug i n the same time pe r i o d . Th is va lue i s i n very c l o se agreement w i th the.vaTue reported by As t r a Pharmaceut ical Products Inc."' (32.5%). (Table X IX ) . Rats have been repor ted to have a h igher drug metabo l i z i ng a c t i v i t y than man [116] . The r e s u l t s obta ined i n the present study seemed to be c on s i s t en t w i th t h i s r epo r t . The observa t i on of an increased percent of i n t a c t drug excreted i n r a t a t a higher dose (20 mg/kg), p o s s i b l y due to s a t u r a t i on o f the metabo l i z i ng enzymes (Table X IX ) , po in t s to the ne ces s i t y f o r c a r r y i n g out k i n e t i c and metabo l i c s tud ies a t .more than one dose l e v e l . - 126 -Table XIX U r i n a r y ' E x c r e t i o n ' o f Toca in ide i n Var ious Species f o l l ow i ng Oral Adm in i s t r a t i o n Species N Dose (mg) % bf .dose Admin is tered Rat 6 20 32.5 Ra t a .4 5,10,15 16.0 Ra t b 4 20 32.32 Guinea P ig 6 20 21.9 6 25 28.9 Dog 1 20 18.5 Monkey 2 25 29.5 2 50 27.8 2 100 37.9 2 200 40.9 2 400 42.8 Man 9-14 40 0-24 hour u r ine samples '"' Values obta ined in present s tudy. A l l o ther va lues obta ined from As t r a Pharmaceut ica l Products Inc . - 127 -I t i s a l s o i n t e r e s t i n g to note t ha t , a s the dose i s i n c r eased , the percent excreted i n t a c t i n u r ine a l so tends to inc rease i n two other spec ies as we l l (guinea p ig and monkeys). N o n - l i n e a r i t y i n e l i m i n a t i o n was observed i n guinea p ig a t a dose o f 25 mg/kg and in monkeys a t 100 mg/kg and above. b) E f f e c t of Dose and Route of Adm in i s t r a t i o n on Ur ina ry Exc r e t i on of Toca in ide Ur ina ry e x c r e t i on s tud ies were c a r r i e d out f o l l ow i n g a dm i n i s t r a t i o n of 5, 10, 15 and 20 mg/kg of t o c a i n i d e a f t e r i n t ravenous , i n t r a p e r i t o n e a l and o ra l routes of a d m i n i s t r a t i o n . The r e s u l t s of these experiments are summarized i n F igure 23. Fur ther exper imentat ion was., c a r r i e d out a t 30 and 40 mg/kg o f t o c a i n i d e admin i s te red i n t r avenous l y ( F i g . 24) . Fo l l ow ing adm in i s t r a t i o n o f 5, 10 and 15 mg/kg o f t o c a i n i d e i n t r a -venously, an average of approx imate ly 16% of the dose admin is tered i s excreted as i n t a c t t o c a i n i d e . There was no s i g n i f i c a n t d i f f e r e n t i n the percent of dose excreted as i n t a c t drug i n 24 hour u r i ne samples f o l l ow i n g o r a l , i n t r a p e r i t o n e a l and in t ravenous routes of a d m i n i s t r a t i o n . B i o a v a i l -a b i l i t y c a l c u l a t i o n s based on th t u r i n a r y ex c r e t i on s tud ies (eq. 3) y i e l d ed va lues c l o s e to.-100% (F igure 25) . X "I00 a f t e r o ra l (or) i n t r a p e r i t o n e a l route B i o ak Y 9 i l - = AJo x 100 (3) a b i l i t y _ -,oo X- a f t e r in t ravenous route This i n d i c a t e s t ha t the drug i s complete ly a v a i l a b l e f o l l ow i n g o ra l :or ; i n t r a p e r i t o n e a l a d m i n i s t r a t i o n . These observa t ions r u l e out the p o s s i b i l i t y o f any f i r s t pass meta-bo l i sm of t o c a i n i d e f o l l ow i n g o ra l or i n t r a p e r i t o n e a l a dm i n i s t r a t i o n i n r a t s . U R I N A R Y EXCRETION O F T O C A I N I D E 60 • i.p. 0 oral • i.v. I 401 D Ot D o a: g 20| W OC (J u w O Q (8) 0 0 F igure 23: (8) (4)(4) (41 P I * 1 0 ro co 1 5 2 0 DOSE IN mg/kg E f f e c t of route of a dm i n i s t r a t i o n on the u r i n a r y ex c r e t i on of t o c a i n i d e in r a t s f o l l o w i n g d i f f e r e n t 1 doses H i n t r a p e r i t o n ea l ^ o r a l I 1 in t ravenous Data presented as mean ± standard dev i a t i o n The numbers i n the .parentheses r e f e r to the number of animals used * I h e V^me? l o r d i f f e r e n t routes '.eft ! 2 0 mgVKg are s i g n i f i c a n t l y d i f f e r e n t (p < 0.01) from tha t a t 5, 10 and 15 mg/Kg - 1 2 9 -10 15 20 25 30 40 D O S E mg/Kg F igure 24: E f f e c t of d i f f e r e n t doses o f t o c a i n i d e admin i s te red i n t r a -venous ly on the u r i n a r y e x c r e t i o n o f i n t a c t t o c a i n i d e i n r a t s . Date presented as mean ± standard d e v i a t i o n . The numbers i n the parentheses' r e f e r :• to the number o f an imals used * S i g n i f i c a n t l y d i f f e r e n t from va lues a t 5, 10, 15 mg/Kg, p < 0.01. • ^ ' 1 0 0 OQ < - J < 5 0 O m 7 / / A T • • • CO o 10 15 20 POSE m 9 / K g F igure 25: % B i o a v a i l a b i l i t y of t o ca i n i de c a l c u l a t ed from u r i na ry exc re t i on data f o l l ow i ng ^ 3 o r a l a n d Q i n t r a p e r i t o n e a l adm in i s t r a t i on of d i f f e r e n t doses. Each of the va lues c a l c u l a t e d from the mean o f 4 to 10 an ima ls . - 131 -Such an obse rva t i on i s c on s i s t en t w i th the human s tud ies where t o ca i n i de was found to be complete ly a v a i l a b l e (100% b i o a v a i l a b i l i t y ) f o l l o w i n g o ra l a dm in i s t r a t i o n [ 18 ] . In t h i s con tex t , t o ca i n i de d i f f e r s from l i d o c a i n e which undergoes ex tens ive f i r s t pass metabol ism. At a dose l e ve l of 20 mg/kg, near l y 40% of the drug was excre ted i n t a c t i n the 24 hour ur ine sample f o l l ow i ng int ravenous a dm i n i s t r a t i o n of t o c a i n i d e . Oral a dm i n i s t r a t i o n o f 20 mg/kg o f t o c a i n i d e r e su l t e d i n the exc re t i on o f 30% of the dose in the u r ine wh i l e 32% of the dose was ex-c re ted i n ' u r ine f o l l ow i n g i n t r a p e r i t o n e a l a d m i n i s t r a t i o n . The reduc t i on i n the .percent of dose excreted i n u r i ne f o l l ow i ng an ora l and i n t r a p e r i t o n e a l route as compared to the int ravenous route may be due to a reduced absorp-t i o n of t o c a i n i d e . 3. Ana l y s i s of the P o s s i b l e Reasons f o r the N o n - l i n e a r i t y in the K i n e t i c s of Toca in ide De r i va t i ons from l i n e a r i t y i n the pharmacokinet ic p r ope r t i e s may r e s u l t from one o r more o f the f o l l o w i n g processes - a b so r p t i o n , d i s t r i b u -t i o n or e l i m i n a t i o n . a) N o n - l i n e a r i t y due to Absorp t ion Processes N o n - l i n e a r i t y i n the abso rp t i on may be due to (1) low i n t r i n s i c s o l u -b i l i t y of the drug, (2) low d i s s o l u t i o n r a t e due to f o rmu la t i on f a c t o r s and/or (3) capac i t y l i m i t e d a c t i v e abso rp t i on processes];'! 1 J]. S ince the drug was given i n t r avenous l y i n the present study, non-1inear behaviour i n the abso rp t i on processes cannot exp l a i n the obse rva t i ons . - 132 -b) N o n - l i n e a r i t y due to D i s t r i b u t i o n a l Processes With regard to drug d i s t r i b u t i o n , d ev i a t i on s from n o n - l i n e a r i t y of dose-concent ra t ion r e l a t i o n s h i p may be due to p ro t e i n b i nd i ng , or t i s s u e b i nd i ng . S ince on ly the f r ee drug i s presumed to be a v a i l a b l e f o r t r a n s -po r t , p r o t e i n b ind ing might con t ro l the r a t e of d i f f u s i o n and hence the e l im i n a t i o n r a t e o f drugs. The degree of b ind ing depends on the drug concen t ra t i on in plasma. P r o t e i n b i nd i ng , which can be adequate ly and.wi th s u f f i c i e n t accuracy desc r ibed by the law of mass a c t i o n , i s an important cause of non - l i nea r dose-concent ra t ion r e l a t i o n s h i p s [117] . N o n - l i n e a r i t y due to plasma p ro t e i n b ind ing i s c ha r a c t e r i z ed by a " d im in i s h i ng steepness of the s lope i n the t ime- log concen t ra t i on data w i th i n c r ea s i ng time va lue and decreas ing concen t ra t i on va lue [118 ] " . The semilog p l o t of plasma concen-t r a t i o n time data d i d not f o l l ow the pa t te rn as desc r ibed f o r a case of n o n - l i n e a r i t y due to p r o t e i n b i n d i ng . Moreover, the outcome of the s a t u r a -t i o n of p r o t e i n b ind ing s i t e s on the PCT curves , i s so dependent upon the exper imental cond i t i ons and the nature o f the drug tha t i t i s not po s s i b l e to draw any conc lus i ons s imply from the PCT curves [119] . I f the pe rmeab i l i t y c h a r a c t e r i s t i c s of the drug are very poor or i f the drug e x h i b i t s one compartment k i n e t i c s and the e l i m i n a t i o n of the drug occurs main ly by metabol ism, s a t u r a t i on of the p r o t e i n b ind ing s i t e s w i l l r e s u l t i n a plasma concen t ra t i on time data as shown i n F igure 26A. The a dd i t i o n a l f r e e drug i n the system w i l l n e i t he r move out nor be meta-bo l i z ed a t a co r respond ing ly inc reased ra te so tha t a concave decreas ing PCT curve may r e s u l t . However, i f the drug possesses a mult icompartment c h a r a c t e r i s t i c s and i s e a s i l y a c c e s s i b l e to a l l the pe r i phe ra l compart:-, ments, s a t u r a t i o n of the p r o t e i n b ind ing s i t e s w i l l l ead to a r ap i d i n i t i a l d e c l i n e so tha t a convex PCT may r e s u l t ( F i g . 26B). t i m e u n i t s F igure 26: Theo re t i c a l p l o t of the e f f e c t of non- l i nea r plasma p ro te i n b ind ing on the plasma l e v e l of t o t a l drug A. Case 1 - drug e x h i b i t i n g one compartment c h a r a c t e r i s t i c s and e l im ina ted main ly by metabolism B. Case 2 - drug e x h i b i t i n g mult icompartmental c h a r a c t e r i s t i c s - 134 -In a d d i t i o n , many drugs are bound to plasma p r o t e i n to a l e s s e r extent i n animals as compared to humans [120] . In man on ly 50% of t o ca i n i de was bound to plasma p r o t e i n . At the dose l e v e l s s tud ied i n t h i s exper iment, s a t u r a t i on of p r o t e i n b ind ing would not be expected to prov ide an answer to the non- l i nea r d i s p o s i t i o n . In a two compartment open model, the plasma h a l f - l i f e i s obta ined V l from 3 (a hybr id ra te constant) which i s a f un c t i on of K-j, l<2 and V*(y—) h , K 2 where K-| i s a t r a n s f e r r a te c o n s t a n t . a n d V 2 are volumes of compartment 1 and 2. I<2 i s the e l i m i n a t i o n r a t e constant V* 2 3 can be expressed mathemat i ca l l y as f o l l ow s : (K^Kg+K^) - / ( K - ^ + K ^ ) 2 - 4 K ^ 2 (4) (5) V* where K , = f - and V* = ^1 -1 "2 I f V-| remained constant and V 2 i nc reased w i th an inc rease i n dose_, then V* and 3 would decrease w i th an" inc rease i n dose[121]. In a two compartment open model, a decrease in the d i s t r i b u t i o n r a t e constant w i th an inc rease in the dose cou ld a l s o cause the s lope of a log blood l e v e l time curve to change - 1.35 -with dose even though the t rue r a t e constant of e l i m i n a t i o n of the drug from the body i s independent of the dose admin i s t e red . Disanto [122] has developed a new gene ra l i z ed non- l i nea r pharmaco-k i n e t i c model, which takes i n to account the non - l i nea r t i s s u e b ind ing of drugs to one or more t i s s ue s a s soc i a t ed wi th each of the two f l u i d compart-ments. In such a model, the c a l c u l a t e d va lues o f K-j, K -|, and show a systemat ic t rend i n r e l a t i o n to dose even though the va lues of A, B and K are held constant and on ly the va lue of C° v a r i e d . The plasma p r o f i l e a l so shows a r ap i d e a r l y d e c l i n e fo l l owed by an exponent ia l decay. Th is model was not c on s i s t en t w i th the plasma p r o f i l e s shown i n F igure 21. Moreover, i f the n o n - l i n e a r i t y was due to d i s t r i b u t i o n a l processes on l y , there would be no change i n the compos i t ion of the u r i n a r y e x c r e t i o n products . However, as w i l l be d i scussed l a t e r , the percent of dose ad-m in i s t e red excreted as i n t a c t compound increased w i th the dose. These preeeeding arguments" i n d i c a t e tha t the n o n - l i n e a r i t y observed i n the present study i s not due to d i s t r i b u t i o n a l processes . c) N o n - l i n e a r i t y due to E l i m i n a t i o n Processes i ) Ex c r e t i on through Kidney - Drugs are e l im ina ted from the body e i t h e r by the process of e x c r e t i o n of unchanged drug and/or by metabo l i c t r ans f o rma t i on . Exc re t i on of i n t a c t drug i n the ur ine can occur e i t h e r by pass ive d i f f u s i o n , by an a c t i v e sec re to ry process or a combinat ion of both processes . Glomerular f i l t r a t i o n and tubu la r r eabso rp t i on f a l l i n t o the category of pass ive d i f f u s i o n and, as such, are dose-independent pro-cesses . A c t i v e tubu la r s e c r e t i o n , on the o ther hand, i s sa tu rab l e a t high concen t ra t i ons of .the drug. - 13.6 -The kidney, c learance in ra t -has been reported to be 1.1 ml/minute [123] . The c a l c u l a t e d c learance of t o ca i n i de i s approx imate ly 0.9 ml/ minute i n d i c a t i n g tha t t h i s compound i s more l i k e l y excreted by g lomeru lar f i l t r a t i o n . (Ac t i ve tubu la r s e c r e t i on fo l l owed by an e f f i c i e n t reabsorp-t i on process can a l s o r e s u l t i n s i m i l a r c learance v a l u e s ) . Under the cond i t i ons of sa tu rab l e tubu la r s e c r e t i o n , a r educ t i on in the f r a c t i o n of i n t a c t drug excre ted w i l l be expected a t h igher s a t u r a t i n g doses. However, i n the present exper imenta t i on , an inc rease i n the percent of the dose admin i s te red excre ted as i n t a c t drug was observed. Hence, s a t u r a t i on of a n - a c t i v e tubu la r s e c r e t i on does not seem to account f o r the non - l i nea r k i n e t i c s of t o c a i n i d e . N o n - l i n e a r i t y i n the e x c r e t i o n k i n e t i c s o f a compound undergoing g lomerular f i l t r a t i o n and t ubu l a r r eabso rp t i on can be brought about by changes i n the u r i n a r y pH. Th is i s a t t r i b u t e d to the f a c t tha t the ex-tent of i o n i z a t i o n depends on the pH and i t i s on ly the un ion ized form o f the drug tha t can d i f f u s e back through the rena l t ubu l e s . The renal c learance of t o ca i n i de was observed to be dependent upon the: u r i na r y pH values i n humans [18 ] . I t was the re f o re necessary to eva luate the i m p l i -c a t i o n o f u r i n a r y pH a l t e r a t i o n s i n the d i s p o s i t i o n o f t o c a i n i d e i n the r a t . Contro l o f the u r i na ry pH va lues cou ld be achieved by the admin is-t r a t i o n of ammonium c h l o r i d e ( a c i d i c pH) and sodium b icarbonate (bas i c pH) to humans [18 ,124] . Maintenance of the ur ine pH i n r a t s was, however, extremely d i f f i c u l t . I t r equ i r e s the use of m u l t i p l e doses of the a c i d i f y i n g , or a l ka ' T i n i z i ng agent. Due to the p o s s i b i l i t y of l o s s of u r i ne samples dur ing anaes thes i a , an o r a l , i . v . or i . p . route of a dm in i s t r a t i o n was not - 137 -p o s s i b l e . However, e f f o r t s were made to in t roduce a cannula i n to the p e r i -toneal c a v i t y and to e x t e r i o r i z e the cannula a t the nape of the neck. The s o l u t i on s used were found to be very i r r i t a t i n g to the r a t and i t was a l s o not po s s i b l e to ma in ta in the pH f o r a s u f f i c i e n t per iod of t ime. A d i r e c t eva lua t i on of t h e . e f f e c t o f pH on the exc re t i on of t o c a i n i d e was the re fo re not po s s i b l e i n the r a t . S tud ies were, however, c a r r i e d out to determine the e f f e c t of d i f -f e r en t doses of t o c a i n i d e on the pH o f u r i n e . Table XX: ' shows the pH va lues of u r i ne samples obta ined from r a t t r ea ted w i th s a l i n e , 10 mg/kg and 20 mg/kg o f t o c a i n i d e . There was no s i g n i f i c a n t d i f f e r e n c e i n the pH va lues a t d i f f e r e n t dose l e v e l s when compared to the c o n t r o l s . Moreover, i f by decreas ing .the pH o f the u r i ne ( a c i d i f i c a t i o n ) the percent of dose excreted unchanged in the u r i ne i n c r eases , i t i s expected to be r e f l e c t e d i n a r educ t i on i n the e l i m i n a t i o n h a l f - l i f e of the com-pound. Such a phenomenon was observed in the case of m e x i l i t e n e , a s t r u c t u r a l l y s i m i l a r an t i a r rhy thm i c agent [124] , Under a l k a l i n e u r i ne pH (pH 8 . 0 ) , 0.6 ± 0.3% of the dose was excreted in the u r i ne and the ty^ °f mex i l i t e ne was 8.6 hours. When the u r i ne pH was mainta ined a t 5, the percent of the dose excreted unchanged in the u r i ne increased to 57.5 ± 11.9 and the t - ^ decreased to 2.8 hours. In c o n t r a s t , i n the present i n v e s t i g a t i o n , a t high doses (20 mg/kg) the percent of dose excreted unchanged i n the u r ine as wel l as the t - ^ va lue increased from that observed a t low doses (5 , 10, 15 mg/kg) ( F i g . 23, 24) . These preceeding f a c t o r s r u l e out the p o s s i b i l i t y of u r i ne pH changes being r e spons i b l e f o r the dose dependency observed i n t h i s s tudy. i i ) B i l i a r y Exc re t i on - With those drugs excreted in b i l e , non-l i n e a r b i l i a r y ex c r e t i on may be an important aspec t , r e qu i r i n g f u r t h e r - 138 -Table XX E f f e c t of D i f f e r e n t Treatments on the Ur ine pH Contro l 6.53a± 0 . 1 9 b ( 8 ) C Sa l i n e 6.58 ± 0.24 (4) Toca in ide 10 mg/kg 20 mg/kg 40 mg/kg 6.67 ± 0.25 6.98 (6.97 6.82 ± 0.22 (5) (4) 6.99) (2) (40 mg/kg and above 6.84 ± 0.18 (4) (100 mg/kg)) a Mean ^ Standard d e v i a t i o n c Number i n the parentheses r e f e r s to .the number o f animals (Test va lues are not s i g n i f i c a n t l y d i f f e r e n t from the con t ro l p < 0.01") - 139 -s tudy. In an e f f o r t to determine the r o l e of b i l i a r y e x c r e t i o n , the faeces samples c o l l e c t e d over a 24 hour per iod were ana lyzed f o r the pre-sence o f the drug. The drug i n the faeces can account f o r the drug unab-sorbed from the stomach ( i n the case o f the o ra l route of adm in i s t r a t i on ) or b i l i a r y and/or s a l i v a r y e x c r e t i on of the drug. Due to the p o s s i b i l i t y of r eabso rp t i on of the drug from the g a s t r o - i n t e s t i n a l t r a c t , the amount found i n the faeces i s l i k e l y to be an underest imat ion of the b i l i a r y and s a l i v a r y exc re to ry processes . Ana l y s i s o f the faeces sample revea led the absence of any i n t a c t or conjugated t o ca i n i de i n the 24 hour faeces samples a f t e r the a dm in i s t r a t i o n of 10 and. 20 mg/kg t o c a i n i d e , i n t r avenous l y . For ex tens ive b i l i a r y e x c r e t i on i n the r a t , there i s a minimum value f o r the molecu lar s i z e (m.wt 325 ± 50), below which l i t t l e b i l i a r y e x c r e t i on of a compound occu r s . In a d d i t i o n , the molecule should possess a s t r ong l y po l a r group which may be a c a r b o x y l , s u l f a t e o r a s u l f o n i c ac id group [125] . S ince t o ca i n i d e has a molecu lar weight of on l y 192,. i t was not expected to be excreted as such i n the b i l e . However, the p o s s i b i l i t y of a conjugated metabo l i t e being excre ted i n the b i l e cannot be complete ly o v e r r u l e d . i i i ) - M e t a b o l i s m - evidence f o r s a t u r a t i on of metabol ism. A number of enzyme systems are i nvo lved i n the b i o t r ans f o rma t i on of drugs. These enzymes are a v a i l a b l e on ly i n f i n i t e q u a n t i t i e s and as such t h e i r c apac i t y may be l i m i t e d , e .g . , g l y c i n e con jugat ing systems [125] and a c e t y l a t i n g system [126] . The b i o t r ans fo rmat i on o f c e r t a i n drugs and chemi - : c a l s cannot be desc r ibed w i th s u f f i c i e n t accuracy by s imple f i r s t order k i n e t i c s . The e l i m i n a t i o n o f s a l i c y l i c a c i d proceeds r e l a t i v e l y s l ow ly as the dose i s inc reased [128] . Th i s i s due to the l i m i t e d capac i t y of - 140 -the enzymes r e spons i b l e f o r the metabol ism o f s a l i c y l i c a c i d . At lower l e v e l s of the drug , when the enzyme concen t ra t i ons are high r e l a t i v e to the drug, the metabo l i c process can be descr ibed by an apparent f i r s t order process . As e a r l y as i n 1948, Boxer and coworkers [129] repor ted what appears to be non l i near k i n e t i c s of e l i m i n a t i o n of s t reptomyc in i n r a t . Dose dependency o f the apparent e l i m i n a t i o n r a te s has been shown f o r o ther compounds such as s a l i c y l i c a c i d [130] , d icoumarol [131] , e thy l b iscoumacetate [132] , probenec id [133] , phenylbutazone [134] and d ipheny lhydanto in [134] . The dose dependent k i n e t i c s of phenylbutazone cou ld be e i t h e r due to subs t ra te i n h i b i t i o n , p r o d u c t i n h i b i t i o n or s a t u r a t i on of the enzyme systems i n vo l v ed . Oxyphenbutazone, a metabo l i t e of phenylbutazone has been shown to i n h i b i t the e l i m i n a t i o n of phenylbutazone i n r a t s [135] . In . the case of d ipheny lhydanto in , 5-(p-hydroxyphenyl j -5 - (pheny lhydanto in ) a major metabo l i t e was found to i n h i b i t the metabol ism both i n v i vo and i n v i t r o [136,137] . In the presence of product i n h i b i t i o n , i t i s l i k e l y tha t the ra te o f d e c l i n e of plasma concen t ra t i on i n the same concen t r a t i on range w i l l decrease w i th an inc rease in dose. S i m i l a r observa t i ons were made in the case o f e l i m i n a t i o n o f d ipheny lhydanto in [136] . Sa tu r a t i on of the enzyme systems r e spons i b l e f o r metabol ism of a drug w i l l l ead to changes i n the termina l h a l f - l i f e of the compound. The f r a c t i o n a l compos i t ion of the ex c r e t i on products w i l l a l s o change w i th dose, due to s a t u r a t i on of c e r t a i n metabo l i c pathways. The f r a c t i o n of dose excreted through the sa tu rab l e metabo l i c pathway decreases w i th i n c r ea s i ng dose. The percent of dose of drug excreted as the i n t a c t drug - 141 -and as the metabo l i t es through non-saturab le pathways w i l l i nc rease w i th the dose. S tud ies were c a r r i e d out to determine the amount of t o c a i n i d e in u r i ne f o l l ow i ng adm in i s t r a t i o n o f . t o c a i n i d e to r a t s : The r e s u l t s are shown in F igure 23, "24. Up to a dose of 15 mg/kg, approx imate ly 15-20% of the dose admin is tered was excreted i n the 24 hour ur ine samples, a time per iod dur ing which e s s e n t i a l l y a l l o f the t o ca i n i d e was e l im ina ted from the an ima l . At a dose of 20 mg/kg, about 38% of the dose admin is tered was excre ted in the 24 hour ur ine sample. A t 30 mg/kg 44 percent and a t 40 mg/kg 51 percent of the dose was excreted as i n t a c t drug. These r e s u l t s suggest tha t the n o n - l i n e a r i t y i n the k i n e t i c s of t o ca i n i de are due to a non - l i nea r e l i m i n a -t i on p rocess . As the percent of dose excreted changes w i th the dose, these r e s u l t s a l s o r u l e out the p o s s i b i l i t y of n o n - l i n e a r i t y due to d i s t r i b u t i o n a l aspec t s . i v ) Add i t i o na l Evidence f o r Sa tu r a t i on o f Metabo l i c Pathways -Fur ther evidence of the ex i s t ence of a sa tu rab l e metabo l i c pathway f o r the e l i m i n a t i o n o f t o c a i n i d e comes from the s tud i e s w i th enzyme induc ing and i n h i b i t i n g agents . Such an approach was used by Dayton and coworkers [134] to demonstrate the involvement of microsomal drug metabo l i z i ng system i n the dose dependent k i n e t i c s of e l i m i n a t i o n of phenylbutazone and i t s ana logs . Pretreatment w i th SKF 525-A inc reased the h a l f - l i f e 8 fo ld* a f t e r 10 mg/kg of phenylbutazone but on ly a 2 ' fo ld '* increase in the h a l f -l i f e was seen a f t e r a 50 mg/kg dose. In the case of d i 'phenylhydanto in, a more marked change in the h a l f -l i f e "of the con t r o l y_s the phenobarbi t a l pretreatment .group was observed_ a f t e r a high dose (50 mg/kg) vs_ a 1 ow dose (20 mg/kg). - 142 -In the present i n v e s t i g a t i o n , u r i na r y ex c r e t i on s tud ies were •carr ied out a t dose l e v e l s of 15 and 20 mg/kg of t o c a i n i d e a f t e r p re-treatment w i th SKF 525-A. SKF 525A pretreatment increased the percent of the dose excre ted as i n t a c t drug ( i . e . decreased the metabolism) a t both dose l e v e l s 15 mg/kg and 20 mg/kg. However, the extent of i n h i b i t i o n was more pronounced a f t e r a dose of 15 mg/kg ( con t ro l 19% vs pretreatment approx imate ly 42%) than a f t e r a .dose, of 20 mg/kg ( con t ro l 38% p r e t r e a t -ment approx imate ly 50%) where non - l i nea r k i n e t i c s were observed. Fu r the r , the a n a l y s i s of the R - r a t i o , a l s o po in t s to the involvement of metabo l i c processes i n the non- l i nea r k i n e t i c s of t o c a i n i d e (Table XXI ) . (See appendix C f o r d e t a i l s on c a l c u l a t i o n . ) I I I . E. METABOLISM 1. Conjugat ion as a Metabo l i c Pathway Conjugat ion r ea c t i on s are one of the important b i o t r ans fo rmat i on processes i nvo l ved i n the e l i m i n a t i o n of exogenous as we l l as endogenous substances. The process o f con jugat ion r e s u l t s i n a metabo l i t e of h igher p o l a r i t y and water s o l u b i l i t y than the parent spec ies and the products formed are u sua l l y r e l a t i v e l y non- tox i c [138] . Conjugat ion can- occur -w i th g l y c i n e , s u l f a t e , g lucu ron i c a c i d , o r n i t h i n e , mercaptur i c a c i d or g lu tamine. Even a c e t y l a t i o n and methy la t i on r ea c t i on s can be cons idered as con jugat ing r e a c t i o n s . G lucuron ic a c i d conjugat ion i s the most widespread and v e r s a t i l e of the con jugat ion r eac t i ons o c cu r r i ng i n man and most mammalian spec i e s , except the ca t [106] . Many f o r e i gn compounds w i th w ide ly d i f f e r e n t molecu lar s t r u c t u r e can bemetabo l i z ed -by t h i s important con jugat ion mechanism. The hydroxyl group of phenols , eno l s , pr imary, secondary and - 143 -Table XXI 'R" r a t i o f o r d i f f e r e n t doses o f t o c a i n i d e Renal C l e a r a n c e 9 Tota l body c l e a r a n c e 5 Renal c l ea rance/ mg/kg Total body c learance [ K e / K E ] = R 5 1.20 7.36 0.16 10 0.87 5.11 0.17 15 0.85 4.79 0.18 20 1.07 2.84 0.38 a Mean va lues obta ined from 3-6 animals a t d i f f e r e n t dose l e v e l s . - 144 -t e r t i a r y a l c o h o l s , carbonyl group of aromat ic and c e r t a i n a l i p h a t i c com-pounds, pr imary and secondary amino-group of c e r t a i n a l i p h a t i c , h e t e r o c y c l i c and aromat ic compounds and t h i o l group of c e r t a i n su lphur compounds undergo g l u cu ron i da t i on [106] . The ready a v a i l a b i l i t y of g l ucu ron i c a c i d fromrr. o ther carbohydrates i n the body accounts f o r the widespread o c c u r r e n G e r , © f g lucuron ide con juga tes . The process o f g lucuron ide con jugat ion can be summarized as f o l l ows [106] •!• pyrophosphorylase glucose-1-phosphate + UTP > UDP glucose + pyrophosphate UDPG-dehydrogenase ' UDP g lucose + 2 NAD + HL > UDP-glucuronic a c i d 1 + 2 NADH + 2H + g lucurony l t r ans f e r a se UDP-gl ucuron ic a c i d + RZH V RZ-gl ucuron ic a c i d + UDP 0 II where Z can be 0, C-0, NH or S. Enzymatic t r a n s f e r of the glucuronyl--moiety of UDP-g-lucuronic .ac id to an amine acceptor has been observed as a metabo l i c pathway f o r a number of amino compounds [139] . 2. Determinat ion of the Presence of Conjugates of Toca in ide The presence of a conjugate can normal ly be i n f e r r e d from the l i b e r a t i o n o f i n t a c t parent compound/metabolite f o l l o w i n g e i t h e r chemical or enzymatic h yd r o l y s i s o f the g lucuronosy l bond. Chemical h yd r o l y s i s i s - 145 -non - spe c i f i c i n tha t i t l i b e r a t e s conjugates of a l l k i nd s . Chemical h yd r o l y s i s u s u a l l y i nvo l ves treatment o f the samples w i th hyd ro ch l o r i c o r su l phu r i c a c i d a t e levated temperatures (100°). Because of the extreme phys i ca l c ond i t i on s used, the aglycones l i b e r a t e d may decompose a t t imes . In some i n s t ances , au to c l av i ng of the samples w i th a c i d a l s o leads to hydro l y s i s of the con jugates . Enzymatic h yd r o l y s i s i s . r a the r s p e c i f i c i n na ture . The enzyme 3-g lucuron idase hydro lyzes the g lucuron ide conjugates; the enzyme a r y l -s u l f a t a s e hydro lyzes s u l f a t e con jugates . However, the enzymes have d i f - . f e r en t a f f i n i t i e s f o r d i f f e r e n t subs t ra tes and the s p e c i f i c i t y of the enzyme a l so d i f f e r s f o r compounds of apparent ly s i m i l a r chemical s t r u c t u r e [140] . Moreover, the enzymes cou ld be po s s i b l y i n h i b i t e d by the presence natura l i n h i b i t o r y substances which p r e - e x i s t or develop dur ing i ncuba t i on [140] . In the present study h yd r o l y s i s experiments were c a r r i e d out us ing both a c i d and enzymes. P r e l im i na r y a c i d h yd r o l y s i s experiments doubled the amount of i n t a c t drug recovered from the ur ine sample, i n d i c a t i n g the presence of near l y 20% of the dose as con jugates . Near ly 6-8% of the dose was shown to be excreted as g lucuron ide conjugate us ing enzymatic hydro lys Th is h yd r o l y s i s mediated by 3-g lucuron idase was complete ly b locked by 1:4 saccharo- lac tone, a very powerful i n h i b i t o r of 3-g lucuron idase [141] . Levvy and coworkers have observed 94-98% of i n h i b i t i o n of the h yd r o l y s i s of 0.01 M'merithyl 3-g lucuron ide when 0.1 M/ saccharo-1:4 lac tone was used [142] . Complete i n h i b i t i o n o f the 3-g lucuron idase mediated h yd r o l y s i s of the conjugate of t o ca i n i de was observed when a s i m i l a r concen t ra t i on o f saccharo 1:4 l a c tone was used i n the present study (Table X X I I ) . The above study thus conf i rms the presence of a g lucuron ide con jugat ing system - 146 -Table XXII E f f e c t of 1:4 SaccharorLactone (0.1 mM ) on Enzyme Hyd ro l y s i s Area r a t i o o f t o ca i n i de d e r i v a t i v e / i n t e r na l standard Contro l Sample w i th enzyme Sample w i th enzyme + i n h i b i t o r 0.28'3 a ±>,0.019b(3)c 0.371 ± 0.009 ( 3 ) d 0.271 - 0.285 (2) a Mean Standard d ev i a t i o n c Number i n the parentheses r e f e r s to the number of samples ^ S i g n i f i c a n t l y d i f f e r e n t from the con t r o l ; ' (pc0 .01 ) - 147 -f o r the e l i m i n a t i o n of t o c a i n i d e i n the r a t . The d i f f e r e n c e observed be-tween the ac i d h yd r o l y s i s and enzyme hyd r o l y s i s may be due to one of the f o l l ow i ng reasons: a) opt imal cond i t i ons f o r the enzymatic h yd r o l y s i s were not ach ieved, or b) conjugates other than a g lucuron ide conjugate may be present , or c) there may be some enzyme i n h i b i t o r present i n the ur ine sample. Add i t i o na l s tud ies were c a r r i e d out to op t im ize the cond i t i ons of enzyme hyd r o l y s i s as we l l as a c i d h y d r o l y s i s . 3. Op t im i za t i on of Enzymatic Hyd ro l y s i s a) E f f e c t of Amount o f Enzyme on the Hyd ro l y s i s of the Toca in ide Conjugate F igure 27 shows the gene ra t i on .o f t o c a i n i d e from the conjugate f o l l ow i ng i n cuba t i on .w i t h d i f f e r e n t amounts of the enzyme. The use of from 0.1 to 0.4 ml. of enzyme (5000 un i t s /m l ) r e su l t e d in no s i g n i f i c a n t d i f f e r e n c e in the y i e l d of t o ca i n i de from the con jugate . In l a t e r experiments 1000 un i t s of enzyme was used f o r g lucuron ide h y d r o l y s i s . b) E f f e c t of Acetate Bu f f e r vs. Hyd roch lo r i c A c i d on the Enzyme Hyd ro l y s i s Use of ace ta te bu f f e r or hyd ro ch l o r i c a c i d to ad jus t the pH o f the incubat ion medium d id not show any marked d i f f e r e n c e in the y i e l d of t o c a i n i d e obta ined from a con juga te , ( con t r o l = 1, d i l u t e hyd roch l o r i c a c i d = 1.64, ace ta te bu f f e r = 1 .66) . (Tab le X X I I I ) . S ince the use of ace ta te bu f f e r cons i de rab l y reduced the time invo lved and the techniques used, l a t t e r s tud ies were c a r r i e d out us ing t h i s bu f f e r system. 3-Oh A M O U NT OF ENZYME [UNITS] F igure 27: E f f e c t of d i f f e r e n t amounts of enzyme on the hyd ro l y s i s of t o ca i n i de conjugate . Data repor ted as mean ± standard d e v i a t i o n . The number i n the parentheses r e f e r s to the number of experiments c a r r i e d out - 149 -Table XXIII E f f e c t o f Acetate Bu f f e r y_s_ Hydroch lo r i c Ac i d Sample Response Ra t i o o f Toca in ide De r i v a t i v e / In te rna l Standard Contro l 0.0899 ± 0 . 0 0 9 a ( 3 ) b With aceta te bu f f e r 0.1698 ± 0.022 (5) With hyd r o ch l o r i c 0.1624 ± 0.013 (4) a c i d a Mean ± standard d ev i a t i o n b Number i n the parentheses r e f e r to the number of samples - 150 -c) E f f e c t o f pH on the Enzyme Hyd ro l y s i s o f Toca in ide Conjugate P r e l im i na r y s tud ies o f the enzyme hyd ro l y s i s of the ur ine samples obta ined from ra t s dosed w i th t o c a i n i d e , were c a r r i e d out at pH 4 .52 , 5.0 and 5.50. There was no apparent d i f f e r en ce s i n the extent o f h yd r o l y s i s at these pH va lues . Depending upon the source of the enzyme, the opt imal pH f o r the enzymatic r e a c t i o n va r i ed between pH 3.40 and pH 7.50 [143] . In the present s tudy, subsequent experiments were c a r r i e d out a t pH 5,00. d) E f f e c t of the Source of Enzyme on the Hyd ro l y s i s of the Toca in ide Conjugate Some o f the r e s u l t s of the experiments c a r r i e d out to determine the e f f e c t o f d i f f e r e n t sources o f enzyme on the h yd r o l y s i s of the con-jugate o f t o c a i n i d e are shown in Table XXIVa. I n i t i a l experiments w i th g lusu l ase d i d not l i b e r a t e any t o c a i n i d e from the con jugates . S i m i l a r r e s u l t s were obta ined whether hyd ro ch l o r i c a c i d was used to ad jus t the pH o f the s o l u t i o n or ace ta te bu f f e r was used f o r ma in ta in ing the pH of the i n cuba t i on medium. In the case of 3-g lucuron idase (mo l l u sk ) , a very poor y i e l d of t o c a i n i d e was obta ined from the conjugate (Table XXIVb). The use o f ® g l u cu r a se s , however, r e su l t ed in c o n s i s t e n t l y h igher y i e l d of the h y d r o l y t i c product and hence was used i n a l l o ther s t u d i e s . Th is study i n c i d e n t a l l y po in ts to the need f o r us ing more than one source o f enzyme i n determin ing the presence of conjugates and i n p a r t i c u l a r when they have to be quan t i t a t e d . e) E f f e c t o f Chloroform on the Hyd ro l y s i s Mediated by 3-g lucuron idase The add i t i o n o f smal l amounts of ch loro form has been repor ted to i nc rease the a c t i v i t y of the 3-g lucuron idase enzyme [114] . However, i n - 151 -Table XXIVa E f f e c t of D i f f e r e n t Source^ of Enzyme on the Hyd ro l y s i s Glucurase vs G lusu lase Enzyme Response a n Contro l 1.0' b 3 ® GIusulase 1.0 3 ® GIucurase 1.82 3 Area r a t i o of t o c a i n i d e d e r i v a t i v e / i n t e r n a l standard Values expressed i n terms of con t ro l = 1 n = number of samples used - 152 -Table XXIVb E f f e c t of D i f f e r e n t Sources of Enzyme on the Hyd ro l y s i s of Toca in ide Conjugate Glucurase vs G lucuron idase (3 ,, ,) mol lusk Enzyme Response n Contro l 1 .00'' 2 ® GIucurase 1 .95 2 GIucuronidase ^ m o l l u s k ) 1 .08 2 Area r a t i o of t o c a i n i d e d e r i v a t i v e / i n t e r n a l standard Values expressed i n terms of con t ro l = 1 n = number of samples used - 153 -our present s tudy , the a dd i t i o n of 10 y l o f ch loro form to the i ncuba t i on medium d id not r e s u l t i n any inc reased y i e l d of the i n t a c t t o c a i n i d e re leased from the conjugate . f ) E f f e c t o f Time o f Incubat ion on the Enzyme Hyd ro l y s i s A l l o f the enzyme hyd r o l y s i s exper iments ! were c a r r i e d out over a per iod of 24 hours. There was no de tec tab le breakdown of t o c a i n i d e under the cond i t i on s used dur ing t h i s p e r i o d . Incubat ion f o r longer than 24 hours d i d not seem to inc rease the extent of h y d r o l y s i s . A l l the l a t t e r experiments were c a r r i e d out over a per iod o f 24 hours. g) Opt imized Cond i t i ons of the Enzyme Hyd ro l y s i s . Bu f f e r - Acetate pH - pH 5.0 Enzyme - Glucurase® Amount o f Enzyme - 1000 un i t s Per iod o f i n cuba t i on - 24 hours 4. Ac id Hyd ro l y s i s a) Ac i d Hyd ro l y s i s o f the Conjugate a t 100° The r e s u l t o f the ac i d h yd r o l y s i s s tud ies are summarized i n F igure 28. Incubat ion o f u r ine samples w i th IN h yd ro ch l o r i c a c i d l i b e r a t e d t o c a i n i d e corresponding to g rea te r than 15% of the dose admin i s t e red . However, when the u r i ne sample was incubated wi th d i s t i l l e d water , about 22% o f the t o c a i n i d e present seemed to have been broken down. Th is sug-gested tha t the es t imat i on of conjugate-made'here i s an underes t imat ion of the amount r e a l l y present i n the u r i n e . When the i n cuba t i on per iod was 2 hours , g rea te r than 50% of the t o ca i n i d e was degraded i n the u r i ne (3) b (3) CONTROL URINE + WATER URINE + A C I D Ac id h yd r o l y s i s of t o ca i n i de conjugate i n oven a t 100°. Data presented a mean + standard d e v i a t i o n . The number i n the parentheses r e f e r to the number of experiments c a r r i e d out . a = one hour incubat ion t ime; b = 2 hour i ncuba t i on time - 155 -sample w i th d i s t i l l e d water. Two hours i ncuba t i on w i th a c i d l i b e r a t e d t o c a i n i d e corresponding to on ly about 8% of the dose adm in i s t e red . Add i t i o na l s tud i e s w i th ac i d s of d i f f e r e n t s t rength and i ncuba t i on per iod of d i f f e r e n t hours p o s i t i v e l y suggested the degradat ion of t o c a i n i d e w i th i n c r ea s i ng t ime o f i ncuba t i on and i n c r ea s i ng s t rength of the a c i d used (Table XXV). b) Ac id Hyd ro l y s i s o f the Conjugate by Au toc l av i ng The use of au toc l av i ng to hydro lyse the conjugate gave r e s u l t s s i m i l a r to the method o f i n cuba t i on a t 100° in an oven ( F i g . 29). The degradat ion of t o c a i n i d e a t t h i s h igher temperature was a l s o ev i den t . Toca in ide degraded to a g rea te r extent a f t e r au to c l av i ng f o r 35 minutes compared to heat ing at 100° f o r one hour. 5. S t a b i l i t y of an Aqueous So l u t i o n o f Toca in ide Hydroch lor ide In view o f the preceeding f i nd ings ,expe r imen ts were designed to study the e f f e c t o f d i f f e r e n t s t rengths of a c i d and d i f f e r e n t per iods of i n cuba t i on on the s t a b i l i t y o f a pure aqueous s o l u t i o n of t o c a i n i d e . The r e s u l t s o f these s tud ies are summarized i n F igure 30, 31. The use o f IN hyd roch l o r i c a c i d f o r 1 hour r e s u l t s i n near l y 17% reduc t i on i n the amount of t o c a i n i d e . The extent of t o c a i n i d e degradat ion inc reased wi th the s t reng th of a c i d used and the t ime o f i n cuba t i on a t 100°. 6. S t a b i l i t y of an Aqueous So l u t i o n of Toca in ide i n the Presence of Added' Urea During these s tud ies i t was observed that t o c a i n i d e tended to de-grade f a s t e r i n the presence of u r ine than i n pure aqueous s o l u t i o n . Th is cou ld probably be due to some mate r i a l present i n the u r i n e . Ur ine - 156 -Table XXV E f f e c t of D i f f e r e n t Strengths of Ac id and du ra t i on of Incubat ion on the Hyd ro l y s i s o"f Toca in ide Conjugate Time 1 hr 2 hr 4 hr 6 hr IN Hyd roch l o r i c Ac id 0.8900 0.9617 - 0.6050 2N Hydroch lo r i c Ac id 0.6308 0.7413 0.571 0.7101 6N Hydroch lo r i c Ac i d 0.6844 0.6924 0.4280 0.3554 •contro l (0.3377) Value expressed are area r a t i o of t o c a i n i d e d e r i v a t i v e / i n t e r n a l standard - 157 -i U J J > < I I UlQC QGQ m a o ~ ] u i O X U J ? § < let i O"to s i a:< < C V J £u_ < ° 0-4 (4)' 0-2 (4) C O N T R O L (3) S A M P L E + W A T E R S A M P L E + A C I D F igure 29: Ac i d h yd r o l y s i s o f t o c a i n i d e conjugate i n an autoc lave samples autoc laved a t 121° f o r 35 minutes w i th water or IN hyd roch l o r i c a c i d . Data represented as mean ± standard d e v i a t i o n . The number i n the parentheses r e f e r s to the number of experiments c a r r i e d out * S i g n i f i c a n t l y d i f f e r e n t from control* (p < O.'oi) 1 0 0 z o & l/t < z 5 0 GO (4) - A A A • • GO -A A A A A • • • a D • • • • • -A A A • • a a a • • a • • A T a a • C O N T R O L A C I D CO 12 N 1 2 N A C I D A C I D A C I D + B L A N K U R I N E F igure 30: S t a b i l i t y o f an aqueous s o l u t i o n of t o ca i n i de a t 100° f o r 1 hour in presence of d i f f e r e n t s t rengths of hyd roch l o r i c a c i d . Data presented as mean o f 2 to 4 experiments as shown in parentheses 1 0 0 • (3) \ n 6 0 h < z U 2 0 (3) rh (3) 1 + 1 (3) rh c n 1 2 T I M E H O U R S F igure 31: S t a b i l i t y o f an aqueous s o l u t i o n of t o ca in i de hydroch lor ide a t 100° f o r d i f f e r e n t per iods o f t ime. Data represented as mean ± standard d e v i a t i o n . The numbers i n the parentheses refer;; to the number o f experiments c a r r i e d out - 160 -samples are r i c h i n urea . Urea i s i n e q u i l i b r i u m wi th cyanate i n an aqueous s o l u t i o n [145] . Pr imary amine groups have been shown to be carbamylated by cyanate s o l u t i o n [146] . So, i t was hypothes ized tha t the presence o f urea i n u r ine cou ld account f o r the f a s t e r degradat ion of t o c a i n i d e in u r ine a t 100° as compared to an aqueous s o l u t i o n of t o c a i n i d e . Experiments c a r r i e d out w i th urea sp iked samples conf i rmed the above hypo thes i s . An aqueous s o l u t i o n of t o c a i n i d e tended to degrade f a s t e r i n presence of added urea . In the presence of 8 M urea < 20% of the o r i g i n a l amount o f t o c a i n i d e remained i n t a c t i n the sample a f t e r i n cuba t i on at 100° f o r 1 hour. The amount of urea used i n the present study was very high compared to what i s present i n the u r i n e . Never the less , t h i s experiment does po in t to the e f f e c t of urea on t o c a i n i d e at e l eva ted temperatures. The deg rada t i on of t o c a i n i d e per se cou ld probably be desc r ibed by a f i r s t order p rocess . The order of the h y d r o l y t i c r e a c t i o n i s , however, not known (cou ld probably be a pseudo f i r s t - o r d e r p r o ce s s ) . conjugated t o ca i n i de > Toca in ide > degradat ion p roduc ts . Because of the complex i ty o f the r ea c t i on i nvo l ved i t was not po s s i b l e to a c cu r a t e l y quan t i t a t e the amount of conjugate present by t h i s method. 7) Presence of Conjugates Other Than Glucuron ide Conjugate Experiments c a r r i e d out even under opt imal cond i t i ons f o r the enzymatic h y d r o l y s i s , cou ld not account f o r a l l o f the t o t a l conjugates observed f o l l ow i ng a c i d h y d r o y l s i s , a s g lucuron ide conjugates of t o c a i n i d e . - 161 -Thus, there seerns to be strong evidence suggest ing the presence of a . conjugate other than a g lucu ron ide . A c e t y l a t i o n i s a known metabo l i c pathway f o r the e l i m i n a t i o n of pr imary amines [106] . P r e l im i na r y work w i th G.C.-M.S. shows some evidence f o r the ex i s tence of an N-acetyl conjugate of t o c a i n i d e i n r a t s . Th is meta-b o l i t e cou ld g i ve r i s e to t o ca i n i d e upon a c i d h y d r o l y s i s . I t i s a l s o po s s i b l e tha t t o c a i n i d e i s e l im ina ted by an as y e t u n i d e n t i f i e d conjugat ing pathway. In t h i s connec t i on , i t i s of i n t e r e s t to note tha t a c i d hydro lysab le conjugates which were ne i t he r g lucuron ide nor s u l f a t e s have been reported as metabo l i t e s of salbutamol [147] and fenoprofen [148] . The pheno l i c metabo l i t e s of imipramine are conjugated through an ac i d s e n s i t i v e bond wi th an unknown substance [149] . 8. Add i t i o na l Me tabo l i t e s of Toca in ide The u r ine samples ex t rac ted under d i f f e r e n t c ond i t i on s were subjected to gas chromatograph mass spec t rometr i c a n a l y s i s under the chromatographic cond i t i ons used f o r the a n a l y s i s o f c y c l i c compounds (exper imenta l-page 75) . a) Dearninated Toca in ide Chromatography of the e x t r a c t obta ined from e x t r a c t i n g of the u r i ne samples a t pH 9.0 and pH 12.0 us ing methylene c h l o r i d e as the so lvent or the e x t r a c t i o n of the f reeze d r i ed u r i ne sample us ing methanol as the so l ven t , y i e l d ed a peak a t 10 minutes i n s i l a r 10-C column. Ana l y s i s of the mass f ragmentat ion pa t t e rn o f t h i s peak revea led the mo lecu la r ion to be 191 m/e. The f ragmentat ion pat te rn obta ined i s shown i n F igure 32. O r i g i n of the compound from t o ca i n i d e was conf i rmed by s tud i e s w i th (S) deuterated t o c a i n i d e . The compound could not be methylated us ing methelute or diazomethane. The proposed s t r u c t u r e of the compound i s shown i n F igure 32. - 162 -CO z LU > I-< -nr 781 Hi Ml F igure 32: Mass^sFe'ct'euitf a rod:" po :stu*at-ed' s t r u c t u r e o f a metabo l i t e of t o c a i n i d e (XV) i n r a t s - 163 -Me tabo l i t e (XVI) - Chromatography of the u r ine e x t r a c t obta ined from the u r ine by d i r e c t e x t r a c t i o n w i th methylene c h l o r i d e or e x t r a c t i o n of the f reeze d r i ed u r ine sample wi th methanol y i e l d e d a peak w i th a r e t en t i on time of 13 minutes. The mass f ragmentat ion pa t te rn i s shown i n F igure 33. The apparent molecu lar ion was a t m/e 218.-Me tabo l i t e (XVII) - Chromatography of the u r i ne e x t r a c t obta ined by e x t r a c t i o n wi th methanol f o l l ow i ng f r eeze dry ing or d i r e c t e x t r a c t i o n w i th methylene c h l o r i d e , y i e l d e d a peak w i th a r e t en t i on time o f 5 minutes. Mass f ragmentat ion pa t te rn of the peak obta ined i s shown i n F igure 34. The mole-c u l a r ion was a t m/e 121. The o r i g i n of t h i s metabo l i t e from t o ca i n i de was conf i rmed by the use o f deuterated t o c a i n i d e . The proposed s t r u c t u r e o f the metabo l i t e i s shown i n F igure 34. 9. I d e n t i f y and O r i g i n of the C y c l i c Compound a) C y c l i c Me tabo l i t e s of Toca in ide Analog Ac i d h yd r o l y s i s o r 3-g lucuronidase h yd r o l y s i s of ur ine-samples from humans r e c e i v i n g t o ca i n i de r e su l t ed i n a s i g n i f i c a n t inc rease ( app rox i -mately 20% of the dose admin is tered) in the concen t ra t i on of f r ee t o c a i n i d e , suggest ing the ex i s tence of a con jugate . B-g lucuronidase mediated hydro-l y s i s was i n h i b i t e d by 1:4 saccha ro - l a c tone , i n d i c a t i n g the conjugate present to be a g lucu ron ide . However, heat or a l k a l i n e treatment of the u r i ne sample y i e l d e d a c y c l i c t o ca i n i de d e r i v a t i v e , 3 - (2 , 6 - xy l y l ) - 5 -me thy l hydantoin (A) [22] which was e x t r a c t ab l e i n to methylene c h l o r i d e a t pH 9.0. Adjustment of pH of the u r i ne sample to 13.0 by a l k a l i n e t reatment , r e s u l t i n g i n the format ion o f a c y c l i c d e r i v a t i v e , p r i o r to a c i d h yd r o l y s i s f a i l e d to generate any t o ca i n i d e i n response to a c i d h y d r o l y s i s . Fu r the r -more, a c i d h yd r o l y s i s or 3-g lucuronidase treatment before the use of the - 1 64 -Figure 33: Mass spectrum of a metabolite of tocainide (XVI) in ra t s . ^ • • •' - 165 -55 z < -J ui t r jJlLilillluJ l l l i u l -y. III 1,1 Figure 34: - Mass spectrum.and pos tu la ted " s t r u c tu re -o f a metabol i te of toca in ide (XVII) in ra ts - 166 -usual e x t r a c t i o n procedure (ur ine to pH 13 .0 , b r i ng back the pH to 9.0 and e x t r a c t w i th methylene c h l o r i d e ) rendered ur ine samples incapab le o f generat ing (A) . The preceeding evidence suggested tha t the c y c l i c compound i s l i k e l y to be a metabonate, r e s u l t i n g from the i n s t a b i l i t y of the g lucuron ide con jugate , under a l k a l i n e c o n d i t i o n s . There i s some evidence suggest ing the ex i s t ence of c y c l i c com-pounds as metabo l i t es of x y l i d i d e l o c a l anaes the t i c s [150,151] . Two c y c l i c metabo l i t es of l i d o c a i n e , which are s ub s t i t u t e d imidazo l i r iones [DliE] .have been i d e n t i f i e d i n human u r i ne [150] ( F i g . 35-B, C ) . The mechanism proposed f o r the format ion of c y c l i c me tabo l i t e B i nvo l ves an i n t r a -molecu lar condensat ion o f a carb ino lamine in te rmed ia te to the n u c l e o p h i l i c amide n i t r ogen . The c y c l i c metabo l i t e s of l i d o c a i n e were shown a l so to.,be produced a r t i f a c t u a l l y from another metabo l i t e of l i d o c a i n e (monoethyl g l y c i n e x y l i d i d e ) when "impure methylene c h l o r i d e " was used f o r e x t r a c t i o n o f u r i ne samples. The presence o f t r a ce amounts o f formaldehyde or acetaldehyde was repor ted to be r e spons i b l e f o r t h i s obse rva t i on . Recent ly ,Morgan and coworkers [151] repor ted two c y c l i c metabo l i t es which are s ub s t i t u t e d im idazo l i d i nones (D,E) and a t h i r d hydantoin meta-b o l i t e (F) of e t i do ca i ne i n man ( F i g . 35) . In s tud ies o f the metabol ism of e t i d o c a i n e , the c y c l i c compound (F) was conf i rmed to be a "me tabo l i t e " , by d i r e c t G.L.C.-mass spec t r o -met r i c a n a l y s i s o f the methanol ic e x t r a c t of f r eeze d r i ed u r ine samples. Th is procedure, c i rcumvents the p o s s i b i l i t y of a metabonate being formed from the a dd i t i o n o f the base to u r ine sample before e x t r a c t i o n , but does not r u l e out the po s s i b l e format ion of hydantoin a t the e levated temperature of the i n j e c t i o n por t of G.C.-M.S. C H r C H 3 ° Figure 35: Structure of the cyclic tocainide derivative (A) and other cyclic metabolites of tocainide analogs. - 168 -These workers proposed a mechanism f o r the fo rmat ion of the sub-s t i t u t e d im idazo l i d i none me t abo l i t e s . However, no s p e c i f i c mechanism was proposed f o r the format ion of the hydantoin me tabo l i t e . The authors s ta ted tha t "a l though there are severa l mechanisms which cou ld account f o r the format ion of the hydanto in , i t i s not po s s i b l e a t t h i s stage to favour any p a r t i c u l a r mechanism" [151] . b) Development o f an A n a l y t i c a l Scheme In t h i s i n v e s t i g a t i o n , i t was des i r ed to study the presence of the c y c l i c compound i n r a t s and a l s o to determine whether t h i s i s i n f a c t i s a "metabo l i t e " or a "metabonate" in t h i s s p e c i e s . An a n a l y t i c a l scheme (Scheme 7) was developed u t i l i z i n g the i n f o r -mation a v a i l a b l e from human s tud i e s a f t e r t o c a i n i d e , l i d o c a i n e or e t i d o -ca ine a d m i n i s t r a t i o n , i n an e f f o r t to answer the f o l l ow i ng ques t i ons . a) Is there a c y c l i c compound excreted i n the r a t u r i n e a f t e r t o c a i n i d e adm in i s t r a t i o n ? b) Is t h i s (A) an endogenous ma te r i a l or i s i t formed from to ca i n i de? c) Is (A) e x t r a c t a b l e a t pH 9.0 and 12.0 us ing methylene c h l o r i d e ? d) Does the a l k a l i n i z i n g agent (sodium hydroxide) used have any e f f e c t on the format ion of (A)? e) Does adjustment of the pH to 9.0 have any e f f e c t on fo rmat ion o f (A)? f ) Does the use of methylene c h l o r i d e i n f l u ence format ion of (A)? g) Is t h i s compound i n f a c t a metabonate formed dur ing e x t r a c -t i o n processes or i s i t a metabo l i t e? h) Is (A) present i n a c i d hydro lyzed u r i ne samples? In an e f f o r t to answer the preceeding ques t i ons , the u r i ne samples ex t ra c ted under d i f f e r e n t exper imental c ond i t i o n s (Scheme 7) were sub-j e c t ed to a n a l y s i s by G.L.C.-mass spect rometry . Scheme 2 Mu l t i p l e Ana l y t i c a l Scheme fo r Determining the Or ig in and Nature of the Cyc l i c Metabol i te URINE SAMPLE (Tocainide/Deuterated toca in ide) © To pH 9.0 © With sodium hydroxide With ammonium ca r -bonate and ammonium hydroxide Mu l t i p l e Ex t rac t i on with methylene ch l o r i de Organic Layer a) b) Aqueous Layer ad jus t pH to 12.5 with sodium hydroxide and hold f o r 1 -1.5 hrs br ing down to pH " 9.0 wi th d i l u t e hydro-c h l o r i c ac id © Di rec t ex t rac t i on of ur ine sample (no pH ad jus t -ments) Mu l t i p l e Ext rac t ions with Methylene Chlor ide Organic Layer Concentrate Mu l t i p l e Ex t rac t ions wi th methylene ch l o r i de Organic l ayer Aqueous l aye r © Freeze drying of ur ine samples Residue d isso lved in methanol and f i l t e r e d © Acid hydro lys i s with 1 ml IN hydroch lor ic a c i d , 100°C for 1 hour in sealed ampoules Concentrate Methylat ion with methyl-8 reagent Acy la t i on with heptaf luorobutyr ic anhydride © Freeze •drying To pH 9.0 with ammonium carbon-ate and ammonium hydroxide Mu l t i p l e ex t rac t ions with methylene ch l o r i de Organic Aqueous layer l ayer a) adjust to pH 12.5 with sodium hydro-xide and hold f o r 1-1.5 hrs. b) bring down the — pH to 9.0 with d i l u t e hydrochlor ide ac id Discard - 170 -G.L.C.-Mass Spectrometry P r e l im i na r y mass spe c t r a l s tud i e s were c a r r i e d out us ing two d i f -f e ren t columns a) 1.8 m x 2 mm i . d . c o i l e d g l a s s column con ta i n i ng 3% OV-17 coated on chromosorb-W(H.P.) (80-100 mesh) b) 1.8 m x 2 mm i . d . c o i l e d g lass column con ta i n i ng 10% S i l a r 10-C. A S i l a r 10-C column was found to g ive maximal separa t i on o f the peaks observed and was used i n l a t e r s t u d i e s . Samples prepared by d i f f e r e n t e x t r a c t i o n procedures (Scheme 7) were i n j e c t e d i n t o the G.C.-mass spectrometer and the f ragmentat ion pat-terns of the compounds were recorded ,on a magnetic tape, as the compounds e l u ted from the column. A f t e r each run , the gas chromatographic t race was recorded on cha r t paper and the mass f ragmentat ion pat te rns of the peaks observed were i n d i v i d u a l l y recorded and ana lyzed . F igure 36 shows a t y p i c a l response obta ined from a G.L.C.-mass spe c t r a l t r a ce o f a methylene c h l o r i d e e x t r a c t of a u r ine sample a f t e r a d j u s t i n g the pH of the u r ine to pH 9.0 w i th sodium hydrox ide . Experiments were c a r r i e d out us ing t o ca i n i d e as we l l as deuterated t o c a i n i d e . Th is helped to d i s t i n g u i s h between the peaks obta ined from endogenous substances and those de r i ved from t o c a i n i d e . F igure 37 shows the chromatogram obta ined when the concentrated methanol ic e x t r a c t of the f reeze d r i ed ur ine was i n j e c t e d i n t o the G.L .C.-mass.spect rometer . d) I d e n t i f i c a t i o n of the S t ruc tu re of the C y c l i c Compound The mass f ragmentat ion pa t te rn of t o c a i n i d e was i n i t i a l l y de te r -mined to f a c i l i t a t e the s t r u c t u r a l a na l y s i s o f the metabo l i t es of i n t e r e s t . F igure 36: G.L.C.-mass spectrometry-G.C. t race of methylene c h l o r i d e ex t r a c t of u r i ne sample us ing SILAR 10-C column. Peak (a) t o c a i n i d e , Peak (b) c y c l i c compound IN5 F igure 37: G.L.C.-mass spectrometry. G.L.C. t race of the methanol ic e x t r a c t of f reeze d r i ed u r i ne samples us ing SILAR 10-C column. Peak (a) t o c a i n i d e , Peak (b) c y c l i c compound - 173 -Add i t i o na l l i t e r a t u r e data were a l so obta ined on the mass spe c t r a l be-^ hav io r of c y c l i c metabo l i t e s of o ther t o c a i n i d e r e l a t e d compounds [150, 151]. The major f ragmentat ion pa t te rn o f t o c a i n i d e i s dep i c ted i n Scheme 8. Toca in ide fragmented p r i m a r i l y by homolyt ic c leavage o f the carbon-carbon bond between the carbonyl and methylene groups to generate the base peak ions a t m/e 44. The o ther peaks observed at m/e 192, 148, 120 and 105 are c on s i s -tent w i th the s t r u c t u r e dep i c ted in Scheme 8. The use of deuterated t o ca i n i d e i n the present s tudy , a l s o helped i n i d e n t i f y i n g the s t r u c t u r e of the unknown compound der i ved from t o c a i n i d e . Peak (b) i n F igure 36, ,37 when subjected to mass spec t r a l a n a l y s i s revea led a parent ion a t m/e 218 ( F i g . 38) suggest ing tha t t h i s compound i s a product from t o ca i n i d e wi th the a d d i t i o n of 26 .mass un i t s t o . t he parent compound. The parent ion (m/e 221) obta ined i n s tud i e s us ing deuterated t o ca i n i d e revea led tha t a l l th ree deuterium ions are i n t a c t i n t h i s "unknown compound". The presence o f ions of the composition-^ CgH g and CgHgN at m/e 105 (108) 2 and 118 (122) r e s p e c t i v e l y , i n d i c a t e s tha t the x y l i d i n e moiety o f t o c a i n i d e i s present i n t h i s compound , (Scheme 9 ) . The presence of a peak at m/e 1.19 (122) and not a t m/e 120 (123) i n d i c a t e s tha t the x y l i d i n e n i t r ogen i s t e r t i a r y . The abundance of m/e 147'(150) i nd i ca te s - tha t^ the . ca rboxy x y l i d i n e moiety of t o c a i n i d e i s a l s o i n t a c t i n t h i s compound, but f o r the l o s s of one hydrogen atom, f u r t h e r suggest ing tha t the amide n i t rogen i s t e r t i a r y and po s s i b l y Numbers i n the bracket corresponds to t r i - d e u t e r a t e d compound. - 174 -Scheme 8 Mass Fragmentation Pattern of Tocainide V \—NH—C—CH—NHo ' C H 3 m/e 192 C H 3 f \ _ N H - ? - C H C H3 m/e 176 CH3 m/e 147 CH3 + C H3 m/e 120 C H 3 ) + CH3 m/e 105 147 1148 218 44 119 105 70 132 U L U J J 219 157 203 F igure 38: Mass spectrum of the c y c l i c compound - 176 -Scheme 9 Mass fragmentation pattern and s t ructure _of_the_c^clic_comgound from tocain ide_ - 177 -par t of a c y c l i c system. The presence of the (m-15) peak a t 203 (206) i n d i c a t e s the presence of a methyl group i n compound ( I ) . The compound ® cou ld be methylated w i th methelute but f a i l e d to form any d e r i v a t i v e w i th hep ta f l uo robu ty r i c anhydr ide . The mass spectrum of t h i s compound was superimposable on the mass spectrum of the "metabonate", 3-(2,6 x y l y l ) , 5-methyl hydantoin obta ined from As t r a Pharmaceut ical P roducts , Inc . us ing a F inn igan model 1015 D quadrapole gas chromatograph-mass spectrometer system (e l e c t r on impact i o n i z a t i o n vo l tage = 2.5 kV a c c e l e r a t i o n p o t e n t i a l , i o n i z a t i o n po t en t i a l = 70 eV) . Fur ther evidence as to the s t r u c t u r e of the compound was obta ined by the company by phys ico-chemica l a n a l y s i s ( i n f r a r e d , proton magnetic resonance spectrum and me l t ing po in t determinat ion) of the compound and by making comparison to the s yn the t i c compounds. e) Me tabo l i t e or Metabonate Scheme 10 summarizes the r e s u l t s of the m u l t i p l e a n a l y t i c a l scheme developed to determine whether the c y c l i c compound i s a metabonate or a me tabo l i t e . A) In step A, the ex i s tence of the c y c l i c compound i n r a t u r i ne was conf i rmed. Th is c y c l i c compound could be o r i g i n a t i n g from an endo-genous mate r i a l or from t o c a i n i d e . Resu l t s of Step B, where deuterated t o ca i n i de was admin is tered to r a t s , conf i rmed tha t the c y c l i c compound i s de r i ved from t o ca i n i d e (the parent ion was 3 mass un i t s heav ier c o r -responding to the three deuter ium ions in the deuterated t o c a i n i d e ) . B) La te r experiments were aimed at determin ing whether I was a metabo l i t e or a metabonate. - 178 -Key to Scheme 10 on Me tabo l i t e or Metabonate 1) S t a r t i n g from the extreme l e f t hand top corner f o l l ow the arrow to a r r i v e a t the f i n a l conc lus ion a t the bottom. 2) An "x" on the arrow i n d i c a t e s tha t t h i s p a r t i c u l a r pathway was not observed in the present s tudy . 3) See d i s c u s s i o n f o r A, B, and C. Scheme- lO.Smnmary datu on the analysis of the origin of the c y c l i c compound No cyclic compound present + cyclic compound 1s not present in the rat either as a metabolite , . (or) as a metabonate. Urine sample from rats dosed with tocainide Urine pH adjusted to >12.0; brought down to pH 9.0 and extracted with methylene chloride CYCLIC COMPOUND -* PRESENT i) may be endogenous/ exogenous 11) may be a metabolite/ metabonate Dose rats with deuterated tocainide LU Ho cyclic com-pound - x -DIRECT extraction of urine sample with methylene chloride; (no pH adjustment) Cycl ic compound observed earlier was probably formed due to the action of alkali used or the impurities present in the solvent CYCLIC . COMPOUND PRESENT Urine sample extracted at pH 9.0 with methylene chloride pH adjusted with a) sodium hydroxide b) ammonium carbonate and ammonium hydroxide Urine pH adjusted to > 12.0; brought back to pH 9.0 and extracted with methylene chloride -X-J Cyclic compound is formed from tocainide Deuterated.cyclic • compound present No cyclic compound No deuterated cyclic com-pound Cyclic compound observed is probably an endogenous one Cyclic compound is probably a metabonate and is formed from an alkaline unstable metabolite at high pHiJ Cyclic compound PRESENT Cyclic compound is not a product of an alka-line unstable metabolite Freeze dry urine sample Dissolve residue in ->methanol & filter — a) conjugates are the sources of * cyclic compound (or) b) cyclic compound is unstable under acidic conditions -Cyclic compound absent Acid hydrolyse urine sample "Freeze dry, dissolve residue <~ 1n methanol, filter Cyclic compound is likely to be metabolite . Cyclic compound Present -) No cyclic compound I Cyclic compound is prob-ably formed due to the presence of some Impuri-ties in methylene chloride I.e. a possible condensa-tion product Cyclic compound Present (conjugates are not the source' of the cyclic metabolite) KO - 180 -i ) E x t r a c t i o n s were c a r r i e d out a t m i l d l y a l k a l i n e pH (pH 9.0) us ing e i t h e r sodium hydroxide or ammonium c a r -bonate or ammonium hydrox ide. Presence of c y c l i c com-pound a t t h i s stage i nd i c a t ed tha t the c y c l i c compound was probably not a product of an a l k a l i n e uns tab le metabo l i t e o f t o c a i n i d e . i i ) Fur ther e x t r a c t i o n s tud i e s wi thout any pH adjustments ru l ed out any p o s s i b l e r o l e o f sodium hydrox ide , ammonium carbonate or ammonium hydroxide in generat ing t h i s compound, i i i ) Experiments c a r r i e d out on the methano l i c e x t r a c t o f f r eeze d r i ed u r i ne ru l ed out the p o s s i b i l i t y of some impu r i t i e s i n methylene c h l o r i d e being r e spons i b l e f o r the product ion o f t h i s c y c l i c compound as was observed i n case of l i d o c a i n e [150] . i v ) Furthermore, t h i s c y c l i c compound was a l s o observed in u r i ne samples which were a c i d hydro l yzed , f r eeze d r i e d and ex-t r a c t ed w i th methanol . Th i s suggests tha t the c y c l i c com-pound i s not l i k e l y formed from a conjugate (an observa t i on con t r a ry to tha t made i n human s tud ies [ 2 2 ] ) . The preceeding evidence seems to suggest tha t the c y c l i c compound observed i s probably not a metabonate and tha t i t i s l i k e l y to be formed in v i v o . However, the r o l e of heat i n generat ing the hydantoin me tabo l i t e from a conjugate of t o ca i n i de was not eva luated due to u n a v a i l a b i l i t y o f the au then t i c me tabo l i t e . The quest ion of the mechanism of fo rmat ion .of ( I ) i n v i vo s t i l l remains to be exp l a i ned . I t i s p o s s i b l e tha t ( I ) may be a me tabo l i t e or a product of a chemical r e a c t i o n , i n v i v o . - 181 -A s t r u c t u r a l l y s i m i l a r hydantoin metabo l i t e accounted f o r 10% of the dose of e t i do ca i ne admin is tered to humans. The c y c l i c compound was denoted as a "metabo l i t e " on the bas i s of i t s presence i n methanol ic e x t r a c t s o f f reeze d r i e d ur ine samples. However, no mechanism was f o r t h -coming from these workers as' to the format ion of the c y c l i c e t i d o ca i ne me tabo l i t e . In the present s tudy , a q u a n t i t a t i v e e s t ima t i on of (A) was not po s s i b l e f o r the f o l l ow i ng reasons: a) Syn the t i c c y c l i c compound was not a v a i l a b l e and hence i t was not po s s i b l e to ob ta in a standard curve f o r q u a n t i t a -t i v e a n a l y s i s . \ 14 3 b) C o r H- l abe l l ed t o c a i n i d e was a l so not immediate ly a v a i l a b l e f o r i n v e s t i g a t i o n . c) Methano l i c e x t r a c t i o n a f t e r f r eeze d ry ing tended to y i e l d more c y c l i c compound compared to methylene c h l o r i d e e x t r a c t s , p o s s i b l y due to the lower s o l u b i l i t y of the c y c l i c compound i n methylene c h l o r i d e . Most o f the t o ca i n i d e seemed to p r e c i p i t a t e out on concen t ra t i ng the methanol ic e x t r a c t of f reeze d r i ed u r i n e . Hence, i t was not even pos s i b l e to ob ta in a rough est imate of r e l a t i v e amounts of t o c a i n i d e and the c y c l i c compound. Pos s i b l e O r i g i n of the C y c l i c Compound In s o l u t i o n , urea e x i s t s i n e q u i l i b r i u m wi th ammonium cyanate [145] NH 0 - C - NH-i^HNCO + NH, 0 - 182 -The time necessary f o r the at ta inment o f e q u i l i b r i u m concen t ra t i on of cyanate from urea depends upon the concen t ra t i on o f u rea , pH, temperature o f p repara t i on o f urea and the temperature o f the s torage [152] . Under moderate c ond i t i o n s , cyanate has been shown to have the a b i l i t y to r eac t w i th amino groups to y i e l d carbamyl d e r i v a t i v e s [153] . The fo rmat ion of carbamates from ammonium carbonate s o l u t i o n was observed to be favoured i n an a l k a l i n e s o l u t i o n [154] . Carbamic a c i d has been shown to decompose extremely r a p i d l y [155] . The carbamyl d e r i v a t i v e s of amino te rmina l res idues i n p r o t e i n molecules have been shown to c y c l i z e i n s t rong a c i d to form hydantoins [155] . O p t i c a l l y a c t i v e carbamyl aminoacids decompose a t t h e i r me l t i ng po in t to form aracemic hydanto ins , as major products [156] . In v iew^of the preceeding i n f o rma t i on , the fol lowing*mechanisms can be suggested f o r the format ion of c y c l i c compound. Mechanism 1 I f the preceeding mechanism i s t r u e , then urea , present i n the u r ine sample cou ld be r e spons i b l e to some extent f o r the fo rmat ion of the c y c l i c compound. Mechanism 2 The second mechanism proposed i nvo l ves the carbamyla t ion of t o c a i n i d e by carbamyl phosphate - a component o f ammonia metabol ism [157] . Ammonia Glutamine carbamyl, phosphate amino compounds ( t o ca i n i de ) <- carbamylated t o ca i n i d e - 183 -Carbamyl phosphate ac ts v i a cyanate in the nonenzymatic carbamylat ion r e a c t i o n . However, i n the c e l l , the phy s i o l o g i c a l carbamylat ing agent i s carbamyl phosphate [158] . The carbamylated t o c a i n i d e c ou l d , i n t u r n , c y c l i z e to y i e l d the c y c l i c compound. Mechanism 3 Carbon d i ox i de e x i s t s i n the body as d i s s o l v ed CC^, b icarbonate and carbamate. Carbon d i ox i de can r eac t w i th un ion ized amino groups to form carbamates [159] /0> — NH - C - CH - NHQ + C0 o -> (5)- NH-C - CH - Nv' V - < ru X — < 1 C - OH CH 3 CH 3 - > C H 3 C H 3 n Since the carbamic ac i d s are very uns tab l e , and the i n t r amo l e cu l a r c y c l i -z a t i on i s a thermodynamical ly favourab le event , a c y c l i c compound may be formed. ^ C H 3 ? ^ / - C C H 3 1 G > NH - C - CH - N —* (0/ NH - C - CH - N = C = 0 CH. ^ ^COOH ^--«- V j H H The presence of r e l a t i v e l y weak l eav ing groups l i n ked to t o ca i n i d e 3 carbaminic a c i d as e s t e r s , y i e l d ed a c y c l i c compound. I t was a l s o noted tha t a dd i t i o n o f dry i c e , sodium hydroxide and methylene c h l o r i d e to 3 t o ca i n i de y i e l d ed the c y c l i c compound in the organ ic phase. L a l k a , D., Personal Communication. - 184 -From the s tud ies c a r r i e d out , the o r i g i n of F cannot be asce r ta ined w i th c e r t a i n t y . However, the a v a i l a b l e evidence suggests tha t i t i s formed i n v i vo i n the r a t . Morgan e t a l . [151] i d e n t i f i e d a c y c l i c metabo l i t e of e t i do ca i ne i n man and suggested tha t t h i s metabo l i c convers ion cou ld be of p o t en t i a l c l i n i c a l s i g n i f i c a n c e . Th is i s based upon the f o l l o w i n g i n f o rma t i on : a) major symptoms of t o x i c i t y due to l o ca l anaes the t i c s are convu l s ions b) hydantoins are we l l known f o r t h e i r a n t i - c onvu l s an t p rope r t i e s c) e t i do ca i ne has lower t o x i c i t y than bup ivaca ine and d) bup ivaca ine has ho' known c y c l i c metabo l i t e Accord ing to Morgan e t a l . [ 151 ] , the lower t o x i c i t y o f e t i do ca i ne when compared to bupivaca ine cou ld po s s i b l y be due to the presence of the hydantoin metabo l i t es of e t i d o c a i n e . I t cou ld be hypothes ized tha t compound ( I ) observed i n r a t a f t e r t o ca i n i de a d m i n i s t r a t i o n , whether formed enzymat i c a l l y or chemica l l y i n v i v o , cou ld conce ivab ly minimize t o ca i n i d e t o x i c i t y . I I I . F. ENZYME INDUCTION 1. Induct ion o f Drug Me tabo l i z i ng Enzymes by Phenobarb i ta l Phenobarb i ta l has a va r i ed a c t i o n on the d i s p o s i t i o n of a number of compounds. Pretreatment w i th phenobarb i ta l has been shown to i n c rease , decrease or have no e f f e c t on the metabol ism ( d i s p o s i t i o n ) of o ther - 185 , -co-admin is tered drugs, depending upon the pretreatment schedule (the dose admin is tered and the du ra t i on of the pretreatment) and the metabo l i c path-way of e l i m i n a t i o n of the co-admin i s te red drug. In a d d i t i o n , pa t i en t v a r i a b i l i t y i s another c o n t r i b u t i n g f a c t o r to the d i f f e r e n t responses observed. The plasma concent ra t i on o f wa r f a r i n has been shown to be i n f l uenced by r egu l a r doses of phenobarb i ta l [ 62 ] . Withdrawal o f co-admin i s te red pheno-b a r b i t a l dur ing wa r f a r i n therapy r e s u l t s i n inc reased plasma concen t ra t i on of w a r f a r i n , l ead ing to po s s i b l e b leed ing compl i e a t i o n s . Add i t i o n of pheno-b a r b i t a l to pa t i en t s on d ipheny lhydanto in therapy has been shown to r a i s e , lower-or e l i c i t nochange in the.plasma concen t ra t i on of the l a t t e r [160] . Th i s i s due to the f a c t t ha t both compounds cause i nduc t i on o f the drug metabo l i z i ng enzymes. Phenobarb i ta l pretreatment r e s u l t s i n a r ap i d decrease i n b i l i r u b i n l e v e l s i n cases o f unconjugated h ype r b i l i r u b i n em i a , , an e f f e c t a t t r i b u t e d to the i nduc t i on of the g lucuron ide con jugat ing pathway [25 ] . P i a f s ky and coworkers f a i l e d to observe any s i g n i f i c a n t changes i n the c learance o f t h eophy l l i n e f o l l o w i n g phenobarb i ta l pretreatment [161] . However, prolonged adm in i s t r a t i o n of phenobarb i ta l has been shown to increase the c learance of t heophy l l i n e i n man [162] . The s t imu l a t o r y e f f e c t o f phenobarb i ta l on the hydroxy lase enzyme system was repor ted to be slow and maximum s t imu l a t i o n was not observed f o r 3-4 days i n . r a t s [ 59 ] . The g lucurony l t r ans f e r a se enzyme system showed no obvious inc rease i n a c t i v i t y u n t i l the t h i r d day of phenobarb i ta l pretreatment [ 23 ] , Phenobarb i ta l br ings about changes i n the d i s p o s i t i o n of co-admin i s te red compounds by more than one mechanism. - 186 -1) Phenobarb i ta l has been shown to inc rease the a c t i v i t y of a number of drug metabo l i z i ng enzymes i n a number of animal spec ies [163,164] . The systemic c learance.-(Cl .) ? o f a" drug undergoing ed iminat ion-by hepat ic .metabol ism can be desc r ibed i n terms of the i n t r i n s i c c learance ( C l j ) o f the drug and the blood f l ow to the l i v e r (Q). where E = hepat i c e x t r a c t i o n r a t i o . When the i n t r i n s i c c l ea rance i s small compared to the l i v e r blood f low the equat ion (1) reduces to C l s = Cl j (7) The va lue of C l j r e f l e c t s the a c t i v i t y of the enzymes r e spons i b l e f o r the metabol ism of the drug. Any inc rease i n the a c t i v i t y of these enzymes (an inc rease i n C l j ) w i l l be r e f l e c t e d i n terms of an inc rease i n the Cl . Prov ided the volume of d i s t r i b u t i o n remains the same, the i n -s crease in c l ea rance w i l l be r e f l e c t e d in terms of a decrease i n the h a l f -l i f e of the drug from the equat ion Cl = K £Vd (8) Kr = ( 9 ) '1/2 Cl = (0.693 Vd) x (10) n/2 - 187 -2) Phenobarb i ta l i nc reases the blood f low to the l i v e r and hence inc reases the d i s p o s i t i o n of h i gh l y c l ea red drugs [165,166] . In equat ion (1) when C l j i s very high compared to Q, the equat ion reduces to C l s = Q (11) which imp l i e s t ha t the systemic c l ea rance o f a drug w i th very high i n -t r i n s i c c l ea rance i s p ropo r t i ona l to the l i v e r blood f l ow . Hence, any inc rease i n l i v e r blood f l ow induced by phenobarb i ta l w i l l r e s u l t i n an increased c l ea rance . I t has been pos s i b l e to c a l c u l a t e the r e l a t i v e c o n t r i b u t i o n of enzyme i nduc t i on and inc reased blood f low induced by phenobarb i ta l on the inc rease in the d i s p o s i t i o n o f a drug [166] . 3) Phenobarb i ta l a l s o has been shown to inc rease the b i l i a r y f l ow and the b i l i a r y e x c r e t i on of both metabo l i zed and non-metabol ized drugs [167,168] . 4) Phenobarb i ta l inc rease the "y" p r o t e i n f r a c t i o n i n the l i v e r l ead ing to inc reased b ind ing (hence inc reased hepat i c uptake) and hence e x c r e t i o n o f bromsulphale in• (BSP) [169] . 5) Phenobarb i ta l can lead to a l t e r a t i o n s i n the p r o t e i n b ind ing p rope r t i e s of drugs r e s u l t i n g i n c l ea rance changes. 6) Compet i t ive i n h i b i t i o n of a metabo l i c pathway may lead to inc rease i n the concen t ra t i on of the other drug [170] . - 188 -7) There i s some evidence to show phenobarb i ta l a l t e r s the d i s t r i b u t i o n a l p r ope r t i e s of some-compounds [171] . Pretreatment of mice or r a t s w i th phenobarb i ta l reduced the t o x i c i t y of l i d o c a i n e [ 63 ] . The r a t e of metabol ism o f l i d o c a i n e in v i t r o , by l i v e r homogenates obta ined from female r a t s p re t rea ted w i th phenobarb i ta l (90 mg/kg g iven i n t r a p e r i t o n e a l l y 48 hours p r i o r to l i d o c a i n e ) was more r ap i d than the con t ro l r a t s . (Test va lues were near l y twice as f a s t as the c o n t r o l s ) . Phenobarb i ta l a l s o s i g n i f i c a n t l y (p < 0.01) inc reased the r a t e o f d e c l i n e o f the b r a i n l e v e l s o f l i d o c a i n e f o l l o w i n g i n t r a p e r i t o n e a l a dm in i s t r a t i o n i n female r a t s [ 63 ] . A s i m i l a r inc rease in the metabol ism and hence a reduced drug l e ve l was seen i n mice as w e l l . Experiments i n dogs have shown tha t i n c o n t r o l s , 25% o f the l i d o -ca ine en te r i ng the l i v e r was removed, as opposed to 50% a f t e r phenobarb i ta l t reatment. The format ion and e x c r e t i o n of g l y c i n e x y l i d i d e , a metabo l i t e of 1 idocaine,was a l so observed f o l l ow i ng phenobarb i ta l t reatment [ 26 ] . These workers c o l l e c t e d on ly two hour u r ine samples and found an inc rease in the quan t i t y o f l i d o c a i n e and two of i t s metabo l i t es a f t e r phenobarb i ta l a d m i n i s t r a t i o n . However, due to the very shor t t ime per iod of u r ine c o l -l e c t i o n , n o conc lus i on cou ld be drawn from these da ta . The plasma l e v e l s of l i d o c a i n e were observed to be lower i n pa t i en t s r e c e i v i n g a n t i e p i l e p t i c drugs, as compared to c o n t r o l s , an obse rva t i on a t t r i b u t e d to the enzyme induc ing p r ope r t i e s o f an t i - c onvu l s an t s [172] . Pa t i en t s who have taken phenobarb i ta l or other p o t en t i a l enzyme inducers over a long per iod of t ime may a l so show somewhat increased to l e rance to the systemic e f f e c t s of cumulat ive doses of. 1 idocaine:.'--. D i s p o s i t i o n of mepivaca ine, another analog of t o ca i n i de was a l so shown to be i n f l uenced by phenoba rb i t a l , both i n mice and r a t s [ 63 ] . - 189 -2. S e l e c t i o n of Dose and Dosing Schedule (phenobarb i ta l ) The dose of phenobarb i ta l and the du ra t i on of pretreatment have an i n f l uence on the extent o f i nduc t i on o f drug me tabo l i z i ng enzymes. Pheno-ba r b i t a l has been shown to have a h a l f - l i f e of about 17 hours in r a t s [173] . I t would requ i r e near l y 4 days (5 h a l f - l i v e s ) to a t t a i n s t eady - s ta te l e v e l s of phenobarb i ta l i n r a t s . S tud ies i n v o l v i n g the e f f e c t of phenobarb i ta l on the d i s p o s i t i o n of o ther drugs have been c a r r i e d out a f t e r doses of pheno-ba r b i t a l ranging from 30 mg/kg/day to 90 mg/kg/day g iven i n t r a p e r i t o n e a l l y f o r 1 to 7 days. New maximal l e v e l s of cytochrome P-450 are achieved w i t h i n two days of pretreatment w i th phenoba rb i t a l . However, i t r equ i r e s near ly three days to ach ieve maximal l e v e l s of cytochrome-bg even a f t e r a s i n g l e dose of phenobarb i ta l [ 5 4 ] . In the present s tudy, i n an e f f o r t to ach ieve maximal i n d u c t i o n , 70 mg/kg of phenobarb i ta l was g iven i n t r a p e r i -t o n e a l l y to r a t s over a 7 day pe r i o d . Add i t i o n a l experiments were a l s o c a r r i e d out a f t e r 3 day pretreatment w i th phenobarb i ta l (70 mg/kg/day). 3. S e l e c t i o n of Dose of Toca in ide Toca in ide e xh i b i t e d non- l i nea r k i n e t i c s i n r a t s a t and above a dose o f 20 mg/kg. A t the 20 mg/kg dose l e v e l the metabo l i c c o n t r i b u t i o n to the t o t a l e l im i n a t i o n was nea r l y 60% as opposed to 84% a t 15 mg/kg. Under s i m i l a r non - l i nea r k i n e t i c s , more marked changes i n the h a l f - l i f e o f d ipheny lhydanto in from the con t ro l to the phenobarb i ta l p re t rea ted group was observed a f t e r l a rge doses (50 mg/kg) compared to lower doses (20 mg/kg) [134] . Hence, i t was decided to c a r r y out the present study a t 20 mg/kg dose l e v e l s . - 190 -4. E f f e c t of Phenobarb i ta l on the D i s p o s i t i o n of Toca in ide a) Plasma Level S tud ies The e f f e c t of phenobarb i ta l on the plasma l e ve l t ime courses of t o c a i n i d e f o l l ow i ng adm in i s t r a t i o n of 20 mg/kg of t o c a i n i d e i s shown in F igure 39. The termina l h a l f - l i f e was s i g n i f i c a n t l y d i f f e r e n t from the con t ro l va lue ( c o n t r o l : - 153 minutes; t e s t 62 m inu tes ) . C a l c u l a t i o n of the area under the plasma l e v e l versus t ime curve (AUC) of t o c a i n i d e a l s o showed a marked d i f f e r e n c e between the con t r o l and t e s t an imals (Table XXVI) . More pronounced d i f f e r en c e s in the AUC va lues were observed a f t e r 7 days of pretreatment w i th phenobarb i ta l when compared to 3 days pret reatment . Pronounced changes in the c l ea rance va lues were a l s o seen a f t e r phenobarb i ta l pret reatment . The changes in c l ea rance cou ld be most ly accounted f o r by changes in the e l i m i n a t i o n i n d i c a t i n g tha t the volume of d i s t r i b u t i o n was not s i g n i f i c a n t l y a l t e r e d by phenobarb i ta l t reatment . b) Ur inary. Exc re t i on S tud ies In an e f f o r t to f u r t h e r c h a r a c t e r i z e the e f f e c t of phenoba rb i t a l , u r i n a r y e x c r e t i o n s tud ies were c a r r i e d out both a f t e r o ra l and in t ravenous adm in i s t r a t i o n of t o c a i n i d e ( F i g . 40 ) . In con t ro l an imals about 38% of the dose o f t o c a i n i d e admin i s te red i s excreted in 24 hour u r i ne samples a f t e r in t ravenous a dm i n i s t r a t i o n of 20 mg/kg t o c a i n i d e . In t e s t an imals on ly 21.9% of the dose i s excreted as i n t a c t drug in the 24 hour u r i ne samples. In the case of the o ra l route of a dm i n i s t r a t i o n even more pronounced e f f e c t s were observed. Contro l an imals excreted about 30% of the dose admin i s te red in 24 hour u r i ne samples as opposed to a con t r o l va lue of 10.3%. S ince the drug has to pass through the l i v e r before reach ing the systemic c i r c u l a t i o n - 191 -1 • ' 120 240 360 480 T I M E (min) F igure 39: E f f e c t o f phenobarb i ta l pretreatment on the plasma concen t r a t i on vs t ime data f o l l o w i n g in t ravenous a d m i n i s t r a t i o n of t o c a i n i d e (20 mg/kg). Data shown as mean ± standard d e v i a t i o n . N = 4 f o r c on t r o l N = 4 f o r t e s t Table XXVI E f f e c t o f Phenobarb i ta l Pretreatment on K i n e t i c s o f Tocain ide Pretreatment (AUC) 24 hr 0 ug.hr.ml~^ Contro l Test Total Body Clearance •1 ml .hr Contro l Test •Volume o f D i s t r i b u t i o n ml Control Test 70mg.kg _ 1 f o r 3 days 22.31' + 3.9 (4) 10.3-17.3 188 253-427 + 31.6 70 mg.kg" 1 f o r 7 days 24.128 10.34 L ± ± 4.182 3.2 (6) (4) 170 + 31 (6) 411 L + 107 (4) 644 + 117 (6) 592 + 154.00 (4) a Values expressed as mean ± standard dev i a t i o n b S t a t i s t i c a l l y s i g n i f i c a n t from con t ro l (p<0.01) d e v i a t i o n , n = number of an imals used a t each data po in t S i g n i f i c a n t l y d i f f e r e n t from con t ro l (p < 0.01) - 194 -f o l l ow i n g o ra l a d m i n i s t r a t i o n , metabol ism can occur to a g rea te r ex ten t a f t e r t h i s r ou te , e s p e c i a l l y f o l l ow i n g i n d u c t i o n . The reduc t i on i n the u r i n a r y l e v e l s of i n t a c t t o c a i n i d e f o l l ow i ng phenobarb i ta l treatment supports the con ten t i on tha t phenobarb i ta l a l t e r s t o ca i n i de d i s p o s i t i o n by i n c r ea s i ng the a c t i v i t y of the metabo l i z i ng enzymes. S ince i t i s known tha t i n humans changes i n u r i n a r y pH b r i ng about changes i n the percent of drug excreted i n t a c t , i t was dec ided to determine whether phenobarb i ta l has any e f f e c t on the u r ine pH va lues i n r a t , thereby b r i ng ing about changes i n the percent of dose excreted as i n t a c t drug. Table XXVII shows the e f f e c t of phenobarb i ta l pretreatment on the u r ine pH va l ue s . There was no s i g n i f i c a n t d i f f e r e n c e between the con t ro l and the t e s t an ima l s . Hence,the observed changes in percent dose excreted cou ld not be accounted f o r by changes in u r i na r y pH va l ue s . c) E f f e c t of Phenobarb i ta l on the Me tabo l i t e Formation A d d i t i o n a l experiments were c a r r i e d out i n an e f f o r t to i d e n t i f y and i f p o s s i b l e , t o quan t i t a t e some o f the metabo l i t e s of t o c a i n i d e , as w i l l be d i scussed i n a l a t e r s e c t i o n . P r e l im i na r y r e s u l t s of the a c i d h yd r o l y s i s experiments used to quan t i t a t e the conjugates of t o c a i n i d e are shown in Table XXVI I I . Even though phenobarb i ta l pretreatment has been shown to induce the g l u cu rony l a t i ng enzymes, the percent of the dose excreted as the a c i d hydro l ysab le conjugate i n the present study was lower f o l l ow i ng phenobarb i ta l pretreatment as compared to the c o n t r o l s . Such an obse rva t i on cou ld be r a t i o n a l i z e d on the bas i s of one of the f o l l ow i ng l i n e s of reason ing . - 195" -Table XXVII E f f e c t o f Phenobarb i ta l Pretreatment On the ur ine :pH val L i e s i n r a t s Treatment n_ P_H Contro l 4 6.58+0.24 ( s a l i n e ) Toca in ide 4 6.67±0.25 (20 mg/kg) Phenobarb i ta l 4 6.40±0.19 (70 mg/kg/day) a Mean ± standard d ev i a t i o n - 196 -Table XXVIII E f f e c t of Phenobarb i ta l Pretreatment on the Ur ina ry Exc re t i on of Ac id Hydro lysab le Conjugate I n t a c t D r u g 9 (as % of dose) Con juga tes 0 (as % of dose) Contro l Rat 1 27.41 28.70 Rat 2 27.87 27.24 Test Rat ,1 5.63 14.08 Rat 2 5.33 15.45 a dose of t o c a i n i d e - 20 mg/kg b as determined by a c i d h yd r o l y s i s a t 100° - 197 -i ) the presence of phenobarb i ta l and/or i t s metabo l i t e i n the ur ine cou ld have a c ce l e r a t ed the degradat ion of t o ca i n i de under the c ond i t i on s used i n the a c i d h yd r o l y s i s s t u d i e s . However, t h i s i s not very l i k e l y , i i ) Phenobarb i ta l has not induced the con juga t ing systems i n the present s tudy, but other enzymatic pathways are i n -duced. Th i s , however i s not very l i k e l y i n l i g h t o f the induc ing a b i l i t y of phenobarb i ta l on the g l u cu rony l a t i ng enzyme systems. i i i ) Phenobarb i ta l has induced the con jugat ing enzyme systems. However, i t has no e f f e c t on the format ion of a carbamic a c i d which i s g l u cu ron i da ted . ( I t has been pos tu la ted tha t i n man t o ca i n i de i s f i r s t converted to a carbamic a c i d which i s then g lucuron ida ted [22]). i v ) Phenobarb i ta l induces the con jugat ing enzyme systems as we l l as other enzymatic pathways. However, the extent of i nduc t i on of o ther enzymatic pathways i s g rea te r than tha t of con jugat ing enzyme systems. The preceeding s tud ies i l l u s t r a t e tha t t o ca i n i de d i s p o s i t i o n i s s u s c ep t i b l e to enzyme induc ing agents ( phenoba rb i t a l ) . I f the i n t a c t drug ( t o ca i n i de ) i s the a c t i v e moiety , c o - adm in i s t r a t i o n of t o ca i n i de w i th an enzyme induc ing agent may r e s u l t i n decreased blood l e v e l s o f t o c a i n i d e . On the other hand, i t might be pos s i b l e to increase, the e l i m i n a t i o n ra te o f t o c a i n i d e w i th the help o f compounds T i k e phenoba rb i t a l . Even though mepivacaine, l i d o c a i n e and t o ca i n i de are c l o s e l y r e l a t e d ana logs , they d i f f e r i n t h e i r route of. metabol ism. Phenobarb i ta l seems to - 198 -a l t e r the d i s p o s i t i o n of a l l three compounds, due to i t s enzyme induc ing p r ope r t i e s . I I I . G. ENZYME INHIBITION STUDIES 1. E f f e c t of SKF 525-A on Drug Me tabo l i z i ng Enzymes Drugs which are metabo l ized by a common pathway are l i k e l y to i n t e r f e r e w i th the metabol ism o f one another . Drugs metabo l i zed by separate routes are not l i k e l y to i n t e r f e r e w i th each o the r , un less the ra te l i m i t i n g c o - f a c t o r or endogenous in te rmed ia tes are shared by both metabo l i c pathways. A knowledge of the extent to which the metabol ism of a compound could be i n h i b i t e d by another agent i s necessary i n order to prevent the po s s i b l e occurrence of unexpected or unwanted e f f e c t s dur ing m u l t i p l e drug therapy. SKF 525-A pretreatment (40 mg/kg, one hour p r i o r to the drug) s i g n i f i c a n t l y enhanced the inc idence o f A - te t rahydrocannab ino l induced m o r t a l i t y i n mice [174] . The LD-50 of v i n b l a s t i n e has been shown to be decreased to a g reat ex tent by SKF 525-A pretreatment [175] . On the other hand, the rapeu t i c advantage i s taken o f the i n h i b i t i n g p r ope r t i e s o f d i s u l f i r a m on the metabol ism of a l coho l [176] . Pretreatment of new born mice w i th SKF 525-A (32 mg/kg subcutaneously one hour before drug) s i g n i f i c a n t l y decreased the m o r t a l i t y due to cyclophosphamide [177] . SKF 525-A, has l i t t l e o r no pharmacologica l e f f e c t of i t s own [178] , but s i g n i f i c a n t l y prolonged the pharmacologica l a c t i o n of o ther drugs [74, 75 ] . Th is was brought about by the i n h i b i t i o n of metabo l i c t rans fo rmat ion - 199 -of these drugs [76 ,77 ] . The p rec i se mechanism by which SKF 525-A i n t e r -f e r e s .w i t h the o x i d a t i v e b i o t r ans f o rma t i on has not been e s t a b l i s h e d . SKF 525-A was suggested to i n h i b i t f a c t o r / f a c t o r s tha t a d i v e r s i t y of drug me tabo l i z i ng enzyme systems possess i n common [ 77 ] . La ter s tud ies have revea led a number of mechanisms r e spons i b l e f o r the p o t en t i a t i n g e f f e c t of SKF 525-A on the pharmacologica l a c t i v i t y of a number of agents . 1) SKF 525-A po ten t i a t e s the a c t i o n of c u r a r i z i n g agents by i n c r ea s i ng t h e i r ac tua l concen t ra t i on a t s p e c i f i c r e cep to r s , by d i s p l a c i n g them from non - spe c i f i c r ecep to rs [179] . 2) Decreases the enzyme concen t ra t i on by i n t e r f e r i n g w i th p ro t e i n syn thes i s or by caus ing dep l e t i on [ 64 ] . 3) Compe t i t i v e l y b locks the drug me tabo l i z i ng enzymes, by competing f o r subs t ra te b ind ing s i t e s (Proca ine) [180] . 4) Non-compet i t i ve ly i n h i b i t s drug me tabo l i z i ng enzymes by non - spe c i f i c i n t e r a c t i o n w i th the enzyme p r o t e i n . ( I n h i b i t i o n of n i t r o r educ t a se [64] and d-demethy lat ion of p - n i t r o an i s o l e [181] ) . 5) Uncouples the TPNH ox i d a t i o n step from drug ox i d a t i o n step -a s p e c i f i c a c t i o n i n d ev i a t i n g the e l e c t r on f low through d i s s o c i a t i n g NADPH consumption and drug o x i d a t i o n [ 64 ] . 6) I n t e r f e r e s w i th pene t ra t i on of subs t ra te through the mic ro-somal membrane [21 ] . 7) Decreases the b i l i a r y exc re t i on of papaverine [182] . However, i t has no e f f e c t on b i l e f low [183] . 8) Changes t i s s u e membrane pe rmeab i l i t y c h a r a c t e r i s t i c s and hence a l t e r s the d i s t r i b u t i o n p rope r t i e s of drugs [184] . - 200 -9) There i s some evidence tha t SKF 525-A a f f e c t s the kidney f unc t i on and hence po s s i b l y e x c r e t i on of some compounds [185] . 10) I t has been shown to decrease the g a s t r o i n t e s t i n a l b lood f low [186] . Th is p o s s i b l y cou ld be expected to decrease the abso rp t i on o f some compounds. The c learance of the t o ca i n i d e ana log , l i d o c a i n e , i n ca t has been shown to be decreased to 60% of the con t ro l va lues f o l l o w i n g SKF adm in i s t r a t i o n (50 mg/kg, i n t r amuscu l a r l y 1 hour p r i o r to l i d o c a i n e adm in i s t r a t i o n ) [187] . The c learance of a drug depends on the e x t r a c t i o n r a t i o and the blood f low to the c l e a r i n g organ. In the case of compounds w i th a very high c l ea rance , the c l ea rance i s p ropo r t i ona l to the blood f l ow to the c l e a r i n g organ. SKF 525-A has been shown to have no e f f e c t on the l i v e r blood f l ow . The decrease i n the c learance va lue of l i d o c a i n e a f t e r SKF pretreatment cou ld be accounted f o r e n t i r e l y by the r educ t i on i n the hepat i c e x t r a c t i o n r a t i o . The d i s p o s i t i o n of l i d o c a i n e was a l s o impaired in mice by SKF 525-A admin is -t r a t i o n ( con t ro l h a l f - l i f e - 7 minutes; SKF 525-A p re t rea ted animals h a l f -l i f e - 40 minutes) [ 27 ] . In v i t r o s tud ies have shown tha t SKF 525-A a l s o i n h i b i t e d the enzyme amidase ( s o l u b i l i z e d ) which hydro lyses Ntethy l g l y c y l x y l i d i d e , a metabo l i t e of l i d o c a i n e [84 ] . Astrom [27] has shown tha t i n v i v o , SKF 525-A (25 mg/kg) had no e f f e c t on the h a l f - l i f e of p r i l o c a i n e , a secondary amine analog of t o c a i n i d e . However, i n v i t r o , S K F 525-A (TO M) i n h i b i t e d the enzymatic h yd r o l y s i s of p r i l o c a i n e by 53% a f t e r 20 minute i ncuba t i on t ime [188] . The D(-) isomer was i n h i b i t e d by 22%. The d i f f e r e n c e i n the r e s u l t cou ld be due to the d i f f e r e n t spec ies o f an imals used i n v i t r o and i n v ivo s tud ies o r - 201 -due to the d i f f e r en c e s i n the amount of the i n h i b i t o r a t the metabo l i z i ng s i t e s i n the two s t u d i e s . Metabo l i c i n h i b i t i o n of s t r u c t u r a l l y s i m i l a r aminoxy l i d ines va r i e s " w ide ly depending upon the nature of the enzyme system respons i b l e f o r the b i o t rans fo rmat i on and the importance of the p a r t i c u l a r metabo l i c pathway on the o v e r a l l e l i m i n a t i o n of the compound. Toca in ide was expected to undergo o x i d a t i v e metabol ism and i t has been shown to be e l im ina ted as a g lucuron ide conjugate both i n man and r a t s . S ince SKF 525-A has been shown to i n h i b i t the o x i d a t i v e b i o t r ans f o rma t i on as we l l as g l u cu ron i da t i on i t seemed l o g i c a l to expect an i n h i b i t i o n of the d i s p o s i t i o n of t o c a i n i d e a f t e r SKF 525-A t reatment . 2. Pretreatment Schedule SKF 525-A exe r t s i t s a c t i o n 15-20 hours f o l l o w i n g a dm i n i s t r a t i o n [189] . Pretreatment t imes rang ing from 0-60 minutes have been used i n prev ious s t u d i e s . In most of the s tud ies SKF 525-A was admin i s te red i n t r a -pe r i t onea l l y , 40 minutes p r i o r to the drug. There i s a lack o f i n fo rmat i on on the k i n e t i c s of SKF 525-A in r a t s . I t was repor ted tha t i n j e c t i o n of 80 mg/kg SKF 525-A i n t r a p e r i t o n e a l l y r e s u l t e d in a concen t ra t i on of 68 yg/kg i n l i v e r t i s s u e , 1 hour a f t e r i n j e c t i o n . The concen t ra t i on decreased r a p i d l y as a f un c t i on of time [ 78 ] . Use of 40 minute pretreatment schedules seemed a l o g i c a l i n view of the absence of any add i t i o na l i n fo rmat ion on the k i n e t i c s of t h i s compound. - 202 -3. S e l e c t i o n of Toca in ide Dose Prev ious experiments have shown tha t t o ca i n i de e x h i b i t s non - l i nea r k i n e t i c s a t and above 20 mg/kg and t h i s phenomenon seemed to be the r e s u l t o f s a t u r a t i o n o f drug me tabo l i z i ng enzymes. A f t e r a 20 mg/kg dose 42% of the dose admin i s te red i s excre ted as i n t a c t t o ca i n i de i n the 24 hour u r ine c o l l e c t i o n . In other words, approx imate ly 58% of the drug was metabo l i zed . At 15 mg/kg dose, about 19% of the drug was excreted as i n t a c t t o c a i n i d e i n 24 hour u r ine samples and approx imate ly 81% of the dose was metabo l i zed . There fore , i f there i s to be an i n h i b i t i o n of the t o c a i n i d e d i s p o s i t i o n i t i s l i k e l y to be more pronounced a f t e r a 15 mg/kg dose- than a f t e r 20 mg/kg. Hence, plasma l e v e l s tud ies were c a r r i e d out a t the 15 mg/kg dose l e v e l . 4. E f f e c t o f SKF 525-A on The D i s p o s i t i o n o f Toca in ide a) Plasma Level S tud ies Shown i n F igure 38 i s the plasma p r o f i l e of t o c a i n i d e f o l l ow i ng int ravenous adm in i s t r a t i o n o f 15 mg/kg dose before and a f t e r pretreatment w i th SKF 525-A. The h a l f - l i f e o f t o c a i n i d e i n con t ro l an imals was app rox i -mately 85 minutes , whereas i n t e s t a n i m a l s t h i s was s i g n i f i c a n t l y increased to approx imate ly 162 minutes. C a l c u l a t i o n o f the t o t a l body c lea rance va lues us ing the r e l a t i o n -sh ip ^ 0 S e C b , - = C learance AUC f J'0 showed a s i g n i f i c a n t reduc t i on i n the c learance f o l l ow i ng SKF pretreatment (Table XXIX) . The change i n c l ea rance cou ld be main ly accounted f o r by the changes i n e l i m i n a t i o n r a t e cons tan t . 1 2 0 2 4 0 3 6 0 4 8 0 T i m e ( m i n u t e s ) F igure 41: E f f e c t o f SKF 525-A pretreatment on the plasma concent ra -t i o n vs time f o l l o w i n g in t ravenous a d m i n i s t r a t i o n of t o c a i n i d e (15 mg/kg). Each curve i s the mean o f 4 an ima l s . Bars represent standard dev i a t i o n s from the mean - 204 -Table XXIX .Ef fect o f SKF 525-A Pretreatment on K i n e t i c s o f Toca in ide —124 hrs 1. AUC 0 ( g/hr/ml) 'Control 10.43±1.43£ (3) Test 16.56±2.lV (5) D i s p o s i t i o n Rate Constant 0.504±0.059 (3) 0.258±0.020c (5) Tota l Body Clearance (ml/hr) 288±35 . (3) 184±23 (5) Volume o f D i s t r i b u t i o n 577±71 (ml) (3) 716±117c (5) 3 S i g n i f i c a n t l y d i f f e r e n t from the con t ro l (p<0.01) b A l l va lues expressed as meantstandard d ev i a t i o n - 205 -A change i n the e l i m i n a t i o n h a l f - l i f e of a compound e x h i b i t i n g mult icompartmental p rope r t i e s can be brought about by changes i n metabol ism ( i n h i b i t i o n of the metabol ism l ead ing to an inc rease i n the ha l f l i f e ) or by changes i n d i s t r i b u t i o n a l c h a r a c t e r i s t i c s . There seems to be some evidence i n the l i t e r a t u r e suggest ing tha t SKF 525-A, in f a c t , does a l t e r the d i s t r i b u t i o n c h a r a c t e r i s t i c s o f c e r t a i n drugs, p o s s i b l y due to changes i n membrane pe rmeab i l i t y [ 184 ] . . b) U r i na ry Exc re t i on Stud ies In order to determine whether the observed inc rease i n h a l f - l i f e was due to changes i n metabol ism or d i s t r i b u t i o n a l p r o c e s s e s , i t was necessary to c a r r y out a dd i t i o n a l s t u d i e s . I f the inc rease i n h a l f - l i f e i s due on ly to d i s t r i b u t i o n a l changes, i t should not have any e f f e c t on the compos i t ion of the u r i na r y ex c r e t i on products . However, i f i n h i b i t i o n o f the meta-b o l i z i n g enzymes i s r e spons i b l e f o r the observed inc rease in h a l f - l i f e , the compos i t ion of the u r i na ry ex c r e t i on product would be expected to change a f t e r pret reatment . Under such cond i t i ons , the percent of dose excreted as i n t a c t d rug 'o r as metabo l i t e whose pathway i s not i n h i b i t e d by pret reatment ,wi 11 increase.^ That percent of dose excreted as a . metabo l i te v i a the pathway tha t i s s u s cep t i b l e to SKF 525-A pretreatment w i l l decrease < a f t e r pret reatment . S tud ies were c a r r i e d out to determine the compos i t ion of u r i n a r y ex c r e t i on products a f t e r 15 and 20 mg/kg dose of t o c a i n i d e g iven o r a l l y o r i n t r avenous l y . F igure 42, 43 shows the r e s u l t s of the u r i n a r y ex c r e -t i o n s t u d i e s . Pretreatment w i th SKF 525-A before o r a l a dm i n i s t r a t i o n of 15 mg/kg dose inc reased the percent of the dose excreted i n ur ine by nea r l y 3 f o l d : > ( con t ro l - 15.9 ± 2.47, Test - 48.49 ± 4 .67 ) . A f t e r a 20 mg/kg dose, a 2 f o l d inc rease was seen in the percent e l im ina ted as 60r 40 PERCENT DOSE EXCRETED INTACT 20 oi EFFECT OF SKF 525-A PRETREATMENT n:6 n=3 TEST DOSE = 15mg.kg* C O N T R O L n=6 C O N T R O L I.V. o r a l T E S T F iaure 42- E f f e c t o f SKF 525-A pretreatment on the u r i n a r y exc re t i on of i n t a c t t o ca i n i de f o l l ow i n g F igure t t t e c t ^ ^ ^ ^ ( ] r m g / k g ) g i v e n Q r a 1 1 y ( o r ) i n t r a V e n o u s l y . Data r ep re -sented as mean ± standard d e v i a t i o n , n = number of animals used S i g n i f i c a n t l y d i f f e r e n t from con t ro l (p < 0.01) EFFECT OF SKF 525-A PRETREATMENT 6 0 n=5 TEST 4 0 K 2 0 CONTROL n--l l ojo o o ok) o o 1° ° lo o Jo o to o o o o o o o o o o o o o DOSE 2 0 0 1 9 / ^ oral F igu re 4 3 : E f f e c t of SKF 525-A pretreatment on the u r i na r y ex c r e t i on of i n t a c t t o c a i n i d e f o l l ow i ng o r a l a dm i n i s t r a t i o n of 20 mg/kg of t o c a i n i d e . Data represented as mean ± standard d e v i a t i o n , n = number of animals used S i g n i f i c a n t l y d i f f e r e n t from the con t ro l (p.< .0.01) - 208 -i n t a c t drug in 24 hour u r i ne samples. As expected, the extent of i n h i b i -t i o n was more pronounced a f t e r a 15 mg/kg dose than a f t e r a 20 mg/kg dose. c) E f f e c t o f Route o f Adm in i s t r a t i o n of Toca in ide on the I n h i b i t i o n of Metabol ism I n h i b i t i o n o f the drug me tabo l i z i ng enzymes w i l l lead to an inc rease in the h a l f - l i f e of a compound. For drugs w i th low c l ea rance there w i l l be l i t t l e change in the a v a i l a b i l i t y of a compound a f t e r o ra l a dm i n i s t r a t i o n when compared to the in t ravenous route f o l l ow i n g enzyme i n h i b i t i o n . In the present case , an almost i d e n t i c a l ex tent of i n h i b i t i o n was observed f o l l o w i n g o ra l a dm in i s t r a t i o n compared to tha t o f in t ravenous adm in i s t r a t i o n ( F i g . 42) Pretreatment o f r a t s w i th SKF 525-A d id not have any e f f e c t on the ur inary , pH va lues (Table XXX). Hence, the d i f f e r en c e in the percent of dose excreted as i n t a c t drug in c o n t r o l s as compared to the t e s t an imals cou ld not be accounted f o r by changes i n u r i n a r y pH. d) E f f e c t o f SKF 525-A on the E l im i n a t i o n of Toca in ide by Rats P re t rea ted w i th Phenobarb i ta l Experiments w i th l i m i t e d number of an imals (n = 3) were c a r r i e d out to determine the e f f e c t of SKF 525-A on the ex c r e t i on of i n t a c t t o c a i n i d e in r a t s p re t rea ted w i th phenobarb i ta l (70 mg/kg/day) . for 7 days. The per-cent of dose excreted as i n t a c t drug in 24 hour u r i ne sample was increased a f t e r SKF 525-A pretreatment ( con t ro l - 20.41 .± 5 .16(5); Test - 3 4 . 1 0 ) . 4 Th i s i s i n agreement w i th the r e s u l t o f Anders and coworkers [ 80 ] , who showed SKF 525-A to be an e f f e c t i v e i n h i b i t o r of enzymes r e spons i b l e f o r the metabol ism of hexobarb i ta l i n phenobarb i ta l p re t rea ted an ima l s . Th i s observa t i on was a t t r i b u t e d to the f a c t t ha t SKF 5250A not on ly i n h i b i t s the induced microsomal enzymes but a l s o the enzymes which are o r i g i n a l l y p resent . 4 Mean ± standard d e v i a t i o n . - 209 -Table XXX E f f e c t of SKF 525-A Pretreatment on the Ur ine pH Value in Rat Contro l - 6.58 ± 0 .24 a (4 ) Test - 6.54 ± 0.26 (8) a Mean ± standard d e v i a t i o n The number i n the parentheses r e f e r s to the number of animals used - 210 -Thus SKF 525-A pretreatment has been shown to a l t e r the metabol ism of t o ca i n i de i n r a t s . I I I . n\. EFFECT OF COMPETITIVE INHIBITION OF METABOLIC ' PATHWAY ON THE DISPOSITION OF TOCAINIDE 1. E f f e c t o f Sa l i c y l am ide on Drug D i s p o s i t i o n Sa l i c y l am i de has been shown not on l y to i n h i b i t the metabol ism of a number of drugs [30 ] 'bu t a l s o has been observed to decrease the absorp t ion o f some drugs [190] . In the present i n v e s t i g a t i o n , we c a r r i e d out a s e r i e s of experiments to determine the i n f l uence of va r i ous pretreatment schedules and route of a dm in i s t r a t i o n of s a l i c y l am i de on the d i s p o s i t i o n of t o c a i n i d e . 2. P repa ra t i on of Dosage Form of Sa l i c y l am ide Sa l i c y l am ide i s poor l y so l ub l e i n water [191] . In order to ob ta i n a s o l u t i o n dosage form o f t h i s drug, i t i s necessary to d i s s o l v e s a l i c y l am i d e i n sodium hydroxide and s l ow ly b r i ng the pH of the s o l u t i o n down to app rox i -mately 9 .4-9.6 us ing d i l u t e hydroch lo r i de a c i d . Because of the appearance of smal l c r y s t a l s a t pH < 9.4, a l l o f the s o l u t i o n s o f s a l i c y l am i de used i n the present study were a t pH 9 .4 -9 .6 . 3. Route of Adm in i s t r a t i o n o f Sa l i c y l am ide and Toca in ide Because o f the a l k a l i n e nature o f t h e - s o l u t i o n used, i t was not po s s i b l e to admin i s te r s a l i c y l am i de i n t r a venous l y . Even very slow i n -travenous i n j e c t i o n (1-2 minutes) of t o c a i n i d e produced arrhythmias i n the animals l ead ing to death w i t h i n a f ewm inu t e s . S ince t o ca i n i d e was - 211 -developed as an o ra l an t i a r rhy thm i c agent, i t was l o g i c a l to admin i s te r t o ca i n i de o r a l l y . To minimize the e f f e c t s , i f any, of the a l k a l i n e v e h i c l e on the abso rp t i on of t o c a i n i d e , an . i n t r a p e r i t o n e a l route o f a dm in i s t r a t i o n was se l ec ted f o r s a l i c y l a m i d e . The outcome of compet i t i ve drug i n t e r a c t i o n s i s more pronounced when the i n t e r a c t i n g spec ies are a t the s i t e of metabol ism at the same t ime. Experiments were c a r r i e d out a f t e r s imultaneous admin i s -t r a t i o n of s a l i c y l am i de ( i n t r a p e r i t o n e a l ) and t o ca i n i de ( o r a l ) . In another exper iment,both s a l i c y l am ide and t o ca i n i d e were admin i s te red o r a l l y a f t e r a 10 minute pretreatment t ime. 4. E f f e c t of Sa l i c y l am ide on the D i s p o s i t i o n o f Toca in ide a) U r i na ry Exc re t i on Stud ies The r e s u l t s o f the u r i na r y e x c r e t i on data are summarized i n Table XXXI. The percent of dose o f t o c a i n i d e excre ted as i n t a c t drug i n the 24 hour ur ine sample was inc reased by nea r l y 2 f o l d f o l l ow i n g a 30 minute pretreatment w i th s a l i c y l a m i d e . In view of the f a c t tha t on ly i n t a c t drug e l im ina ted i n u r i ne was measured,the observed r e s u l t s cou ld be p o s s i b l y due to (a) an inc rease in the absorp t ion of t o c a i n i d e f o l l ow i ng pret reatment , (b) a decrease i n the extent of metabolism ( po s s i b l e due to a conjugated s p e c i e s ) , (c) a s imultaneous inc rease i n abso rp t i on and a sma l l e r ex tent of decrease in the metabol ism or (d) a s imultaneous decrease in the absorp t ion and a g rea ter extent of decrease i n the metabol ism. Prev ious s tud ies on the k i n e t i c s o f t o c a i n i d e have shown t o ca i n i d e to be almost complete ly absorbed a t 5, 10 and 15 mg/kg doses. Hence an a l t e r a t i o n i n absorp t i on i s not a l i k e l y exp l ana t i on f o r the observed r e s u l t s . Sa l i c y l am ide i s more l i k e l y t o ' i n h i b i t the metabol ism o f - 212 -Table XXXI E f f e c t o f Sa l i c y l am ide on Toca in ide Exc r e t i on Study Number 1 Sa l i c y l am i de Pretreatment Schedule 30 minutes p r i o r to t o ca i n i de dosing Route of Adm in i s t r a t i o n Sal icy1 amide C o n t r o l 0 Test i n t r a -per i toneal Toca in ide ORAL ORAL % dose excreted as i n t a c t t oca in -ide i n 24 hour u r ine sample .16o20 ±3.70 31.06 ± 6.70' 10 minutes p r i o r to t o ca i n i de dos ing Contro l Sharn^ Test o ra l ORAL ORAL ORAL 8 6 16.20 ± 3.70 24.49 ± 1.96 e 28.60 ± 5.43 e , f s imultaneous adm in i s t r a -t i o n w i th t o ca i n i d e Contro l Sham Test i n t r a -pe r i t onea l ORAL ORAL ORAL 16.20 ± 3.70 18.48 ± 2.40 24.31 ± 2.80 e' f a Dose of t o ca i n i d e hydroch lo r i de 15 mg/kg expressed as base D N r e f e r s to the number of animals used c Con t ro l s had no pretreatment d Sham - t r ea ted w i th the v e h i c l e on ly e S t a t i s t i c a l l y s i g n i f i c a n t from con t ro l (p < 0 , 01 \ f S t a t i s t i c a l l y not s i g n i f i c a n t from sham A l l va lues expressed as mean ± standard d ev i a t i o n - 213 -t o ca i n i d e by a compet i t i ve mechanism. S imultaneous-decrease in abso rp t i on and an i n h i b i t i o n of the metabol ism to a r e l a t i v e g rea te r extent cou ld a l s o , i n theory , y i e l d s i m i l a r r e s u l t s . Under the p resent exper imental des ign i t . -was not po s s i b l e to d i s t i n g u i s h between these two mechanisms. Simultaneous adm in i s t r a t i o n of t o ca i n i de and s a l i c y l am i de r e s u l t e d i n a marginal but not s i g n i f i c a n t i n c rease i n the percent o f i n t a c t drug excreted i n 24 hour ur ine sample. Oral a dm in i s t r a t i o n of s a l i c y l a m i d e , 10 minutes p r i o r to t o c a i n i d e , inc reased the percent of dose excre ted as i n t a c t drug i n 24 hour s i g n i f i -c an t l y as compared to the c o n t r o l s . However, t h i s inc rease was not s i g n i f i c a n t l y d i f f e r e n t from the sham t r ea ted an ima l s . Th is r e s u l t po i n t s to the f a c t tha t o ra l a dm i n i s t r a t i o n of an a l k a l i n e s o l u t i o n in i t s e l f causes some i n h i b i t i o n of the metabol ism o f t o ca i n i de i n the r a t . There seems to be a t rend i n d i c a t i n g a p o s s i b l e i n h i b i t i o n o f meta-bo l i sm of t o ca i n i de f o l l ow i ng s a l i c y l am i de a d m i n i s t r a t i o n . S i n c e , l e s s than 10% of the dose of t o c a i n i d e i s excreted as the g lucuron ide conjugate i n r a t s compared to a va lue o f g rea te r than 20% i n man, s a l i c y l am i de i s more l i k e l y to have a profound e f f e c t on d i s p o s i t i o n o f t o ca i n i de i n man than i n r a t s . Fur ther s tud ies are warranted i n man to determine the s i g n i f i c a n c e o f t h i s p o s s i b l e drug i n t e r a c t i o n , e s p e c i a l l y i n l i g h t of the l i m i t e d capac i t y of t h i s p a r t i c u l a r metabo l i c pathway and tha t a number o f compounds (drug and endogenous substances) share t h i s pathway of e l i m i n a t i o n . - 214 -SUMMARY AND CONCLUSIONS 1. A s e n s i t i v e and s p e c i f i c E .C.D.-G.L .C. a n a l y s i s capable of measuring picogram q u a n t i t i e s of t o ca i n i de in b i o l o g i c a l f l u i d s was developed. Th is method i s based on the convers ion of t o ca i n i de to a hep ta f l uo ro -buty ry l d e r i v a t i v e by d e r i v a t i z i n g t o ca i n i d e w i th hep ta f l uo robu ty ry l anhydr ide a t 55° f o r 50 minutes in hexane. 2. The bas ic pharmacokinet ic p r ope r t i e s of t o c a i n i d e i n r a t were s tud ied a t dose l e v e l s of 5, 10, 15, 20 and 40 mg/kg o f t o c a i n i d e . Toca in ide was found to be complete ly absorbed over the dose range of 5-15 mg/kg. Toca in ide e xh i b i t e d non- l i nea r k i n e t i c s of d i s p o s i t i o n a t and above a dose of 20 mg/kg. The n o n - l i n e a r i t y was ev ident from the d i sp ropo r -t i o na t e increase in the area under the plasma l e ve l t ime curve w i th inc rease i n the dose admin i s t e red . A g rea te r than 10% inc rease i n the percent of dose excreted as the i n t a c t drug in the u r i ne a t and above doses of 20 mg/kg suggested the presence of a sa tu rab le metabo l i c pathway f o r the e l i m i n a t i o n of t o ca i n i de in r a t . 3. S tud ies of the metabol ism of t o ca i n i de revea led the presence of appro-x ima te l y 20% of the dose as a c i d hydro lysab le conjugate in the u r i n e . For ty percent of t h i s conjugate was i d e n t i f i e d as the g l u cu ron ide , f o l l ow i ng hyd ro l y s i s w i th 6 -g lucuron idase. The hyd r o l y s i s mediated by B-g lucuronidase was blocked by i t s s p e c i f i c i n h i b i t o r , 1:4 saccharo-l a c t one . In a d d i t i o n , small q u a n t i t i e s of a c y c l i c hydantoin d e r i v a t i v e o f t o c a i n i d e , a deaminated product of t o ca i n i de and an a ce t y l a t ed t o ca i n i d e were a l so found to be present as metabo l i t e s from G.C.-mass spec t r a l s t u d i e s . - 215 -A m u l t i p l e a n a l y t i c a l scheme was developed to determine the presence and to i d e n t i f y the o r i g i n o f the c y c l i c d e r i v a t i v e of t o ca i n i de i n r a t . The c y c l i c hydantoin d e r i v a t i v e which was repor ted to be a "metabonate" i n humans was in f a c t a metabo l i t e i n r a t s . Pretreatment o f the r a t s w i th phenobarb i ta l markedly reduced the h a l f -l i f e of t o c a i n i d e . I t a l s o r e su l t ed i n a s i g n i f i c a n t decrease in the percent of dose excreted as the i n t a c t t o c a i n i d e . The i nduc t i on was more pronounced f o l l ow i ng o ra l a d m i n i s t r a t i o n . There was a reduc t i on i n the amount of a c i d hydro lysab le conjugate excreted in the u r ine f o l l ow i ng phenobarb i ta l pret reatment . Th is suggests tha t the other pathways of e l i m i n a t i o n o f t o ca i n i de are probably induced to a g rea te r extent than the con jugat ing pathways. SKF 525-A pretreatment markedly prolonged the h a l f - l i f e of t o c a i n i d e . The i n h i b i t i o n of t o ca i n i de metabol ism was r e f l e c t e d i n terms o f the increased percent of the dose being excreted as the i n t a c t drug i n the u r i n e . A g rea te r extent of i n h i b i t i o n of the metabol ism by SKF 525-A was observed f o l l ow i n g 15 mg/kg dose than a f t e r 20 mg/kg o f t o c a i n i d e . Th is f u r t h e r supports the ex i s tence of a sa tu rab le metabo l i c pathway f o r the e l i m i n a t i o n of t o c a i n i d e i n r a t s . Pretreatment of the animals w i th a compet i t i ve i n h i b i t o r of the g lucuron ide con jugat i ve pathway ( s a l i c y l a m i d e ) , r e s u l t e d in increased ex c r e t i on of the i n t a c t drug i n the u r i n e . However, the extent of i n h i b i t i o n was dependent upon the route of a dm in i s t r a t i o n as wel l as on the pretreatment schedule used. The d i s p o s i t i o n of t o ca i n i de seems to be su s cep t i b l e to the e f f e c t of enzyme i n h i b i t i n g agents in r a t s . -216 -The r e s u l t s obta ined i n the present study po in t to the ne ces s i t y f o r c a r r y i ng out s i m i l a r experiments in humans so that the pa t i en t s cou ld ob ta in maximum bene f i t from t o ca i n i d e therapy. - 217 -REFERENCES 1. Howi t t , G. , "Drug con t ro l o f c a rd i a c a r rhy thm ias " . In Jones, A. M. (ed.) "Modern trends i n c a r d i o l o g y " . London Butterworths (1969) p. 343. 2. Fakkro, A. M., R i t c h i e , R. F. and Cown, B., "Lupus l i k e syndrome induced by Proca inamide" . Am. J . C a r d i o l . 20: 367-373 (1967). 3. Southworth, J . L., McKusick, V. A . , P i e r c e , E., and Kawson, L. F., " V e n t r i c u l a r f i b r i l l a t i o n p r e c i p i t a t e d by ca rd i a c c a t h e t e r i z a t i o n - , complete recovery of the pa t i e n t a f t e r f o r t y - f i v e minutes" . JAMA 143: 717-720 (1950). 4. Giane- l ly , R. E., Groeben, J . 0 . , Sp ivack , A . , and Ha r r i s on , D. C , " E f f e c t o f l i d o c a i n e on v e n t r i c u l a r arrhythmias i n pa t i en t s w i th coronary heart d i s ea se " . N. Eng. J . Med. 277: 1215-1219 (1967). 5. Yeh, B. K. and Go s s e l i n , A. J^., "Current s t a tus of an t i - a r r hy thm i c drugs" . Rat ion.Drug Ther. -9: 1-6 (1975). 6. Moss, A. J . and Pa t ton , R. D., "An t i a r rhy thmic agents " . Char les C. Thomas. S p r i n g f i e l d 1973, p. 50. 7. Boyes, R. N., S co t t , D. B., Jebson, P. J . , Godman, M. J . and J u l i a n , D. G. , "Pharmacok inet ics of l i d o c a i n e i n man". C l i n . Pharmacol. Ther. 12:. 105.-116 (1971). 8. Ha r r i s on , D. C , Stenson, R. E. and Cons tan t ino , R. T . , "The r e l a t i o n -sh ip of blood l e v e l s , i n f u s i o n r a t e and metabol ism of l i d o c a i n e to i t s an t i a r rhy thm i c a c t i o n " . In Sandol E. F l ens ted- Jensen , E., and G lesen, K. H. (eds.) "Symposium on ca rd i a c a r rhy thm ias " . (1970) p. 427-444. 9. Boyes, R. N., Adams, H. J . and Duce, B. R., "Oral abso rp t i on and d i s -p o s i t i o n k i n e t i c s of l i d o c a i n e hydroch lo r ide i n dogs". J . Pharmacol. Exp. Ther. ]7±: 1-8, (1970). 10. Boyes, R. N. and Keenaghan, J . B., "Some aspects of the metabolism. and d i s t r i b u t i o n of l i d o c a i n e i n r a t s , dogs and man". In S c o t t , D. B. and J u l i a n , D. G. ( e d s . ) , " L i doca i ne i n the treatment o f v e n t r i c u l a r a r rhy thmias " . Proceedings of a symposium held i n Edinburgh i n September 1970. Edinburgh and London F\ and>.S.. L i v i ngs t one (1971). p. 140-151. 11. S t rong , J . M., Pa rker , M., and A t k i n son , A. J . J r . , " I d e n t i f i c a t i o n of g l y c i n e x y l i d i d e i n pa t i en t s t r ea ted w i th int ravenous l i d o c a i n e " . C l i n . Pharmacol. Ther. 14: 67-72 (1973). 12. S t rong , J . M. and A t k i n s on , A. J . J r . , "Simultaneous measurement o f plasma concen t ra t i on of l i d o c a i n e and i t s deethy la ted metabo l i t e by mass fragmentography". Ana l . Chem. 44: 2287-2290 (1972)'. - 218 -13. S t rong , J . M., May f i e l d , D. C , A t k i n s on , A. J . J r . , Bu rn ' s , B. C. and Raymon, F., "Pharmacolog ica l a c t i v i t y , metabol ism and pharmaco-k i n e t i c s of g l y c i ne x y l i d i d e " . C l i n . Pharmacol. Ther. 17: :184-194 (1975). 14. H a l k i n , H., M e f f i n , P., Melmon, K. L., Rowland, M., " In f l uence o f conges t ive heart f a i 1u r e on blood l e v e l s of l i d o c a i n e and i t s a c t i v e mono deethy la ted me t abo l i t e " . C l i n . Pharmacol. Ther. 1_7: 669-676 (1974). 15. Duce, B. R., Smith E. R., Boyes, R. N., and Byrnes, E. N., "The acute an t i a r rhy thm i c and ' toxic e f f e c t s of f 2-amino-2 1 ,6" 'prop ionoxyl i d i d e (W-36095) i n mice and dogs". The Pharmaco log is t .•15: 192 (1973) [ A b s t r . ] 16. C o l t a r t , J . D., Berndt , T. B., Kerno f f , R. and Ha r r i s on , D. C , "An t i a r rhy thmic and c i r c u l a t o r y e f f e c t s of As t ra W-36095. A new l i d o c a i n e - l i k e agent" . Am. J . C a r d i o l . 34: 35-41 (1974). 17. Wink le , R. A . , Me f f i n , P. J . , F i t z g e r a l d , J . W. and Ha r r i s on , D. C , " C l i n i c a l e f f i c a c y and pharmacok inet ics o f a new o r a l l y e f f e c t i v e an t i a r rhy thm i c t o c a i n i d e " . C i r c u l a t i o n . 54: 884-889 (1976). A. 18. L a l k a , D., Meyer, M. B., Duce, B. R.v-and E l v i n , A. T . , " K i n e t i c s of the o r a l an t i a r rhy thm i c l i d o c a i n e congener, t o c a i n i d e " . C l i n . Pharmacol. Ther. 19: 757-766 (1976). 19. Shand, D. G . , M i t c h e l l , J . R., and Oates, J . A . , "Pharmacok inet ic drug i n t e r a c t i o n s " . In G i l l e t t e , J . R. and M i t c h e l l , J . R., "Handbook of exper imental Pharmacology". 28: 272-344.; (1975). 20. Conney, A., H. "Pharmacolog ica l imp l i ea t i cms of microsomal enzyme i n d u c t i o n " . Pharmacol. Rev. 19: 317-366 (1967). 21. Mannering, G. J . , " S i g n i f i c a n c e o f s t i m u l a t i o n and i n h i b i t i o n of drug metabol ism i n pharmacologica l t e s t i n g " . In Beurger, A. (ed.) "Se lec ted pharmacologica l t e s t i n g methods". VO1T3. Mercel Dekker, N.Y. (1968) p. 51-119. 22. E l v i n , A. T . , Keenaghan, J . B., Byrnes, E. W., Tenthorey, P. A . , McMaster, P. D., Takman, B. H., L a l k a , D., Manion, C. V., Baer, D. T. , Wo lsh in , E. M., and Meyer, M. B., "Conjugat ion o f t o ca i n i de i n man: Evidence f o r a novel pathway of b i o t r ans fo rmat i on f o r a pr imary amine". The Pharmacolog is t 18: 114(1976) [ A b s t r . ] 23. Ze idenberg, P., O r ren ius , S . , E r n s t e r , L., " Increase i n l e v e l s of g l u cu r ony l a t i ng enzymes and as soc i a t ed r i s e i n a c t i v i t i e s of m i to -chondr i a l o x i d a t i v e enzymes upon phenobarb i ta l a dm i n i s t r a t i o n in the r a t " . J . C e l l . B i o l . 32: 528-531 -(1967) . 24. Lacamwasm, D. S . , F r a n k l i n , C. and Turner, P., " E f f e c t of phenobarbitone on hepat i c drug metabo l i z i ng enzymes and u r i na r y D - g l u c a r i c a c i d ex c r e t i on i n man". Br . J . C l i n . Pharmacol. 2: 257-262 (1975). - 219 -25. Y a f f e , S. J . , Levy, G. , Matsuzawa, T . , and B a l i a h , T . , "Enhancement of g lucuron ide con juga t ing c apa c i t y i n a hyperb i1 i rub inaemic i n f a n t due to apparent enzyme i nduc t i on by phenoba rb i t a l " . N. Eng. J . Med. 275: 1461-1466 (1966). 26. D i f a z i o , C. A. and Brown, R. E., "L idoca ine metabol ism i n normal and phenoba rb i t a l - p re t r ea ted dogs". Anaes thes io logy . 36: 238-243 (1972). 27. Astrb'm, A . , "Fac tors determin ing a c t i o n and t o x i c i t y of l o c a l anaes thes t i c s as i l l u s t r a t e d by exper imenta l r e s u l t s w i th some x y l o c a i n e - - l i k e compounds". In 3rd Congressusmundia l i s anaesth-s i o l o g i e V o l . 1, p. 306-310 (1964). 28. Levy, G. and Matsuzawa, T . , "Pharmacok inet ics of s a l i c y l am i de e l i m i n a -t i o n in man". J . Pharmacol. Exp. Ther. 256: 285-293 (1967). 29. Ba r r , W. H., "Some physiochemical and phy s i o l o g i c a l f a c t o r s i n i n t e s t i n a l drug abso rp t i on and metabo l i sm". Ph.D. D i s s e r t a t i o n , Univ. o f C a l i f . , San F r anc i s c o , 1967. 30. Levy,G. and P r o ckna l , J . A . , "Drug b i o t r ans fo rmat i on i n t e r a c t i o n s i n man I . Mutual i n h i b i t i o n o f g lucuron ide fo rmat ion of s a l i c y l i c a c i d and s a l i c y l am i de i n man". J . Pharm. S c i . 57: 1330-1335 (1968). 31. Levy, G. and Yamada, H., "Drug b i o t r ans f o rma t i on i n t e r a c t i o n s i n man I I I . Acetaminophen and s a l i c y l a m i d e " . J . Pharm. S c i . 60: 215-221 (1971). 32. Morgan, D. J . , Smyth, M. P., Thomas, J . and V ine , J . , " C y c l i c meta-b o l i t e s of e t i doca i ne in humans". Xenob io t i c s 7_: 365-375.(1977). 33. McDev i t t , D. G. , N i e s , A. S . , W i l k i n son , G. R., Smi th , R. F., Woosley, R. L. and Oates, J . A . , "An t i a r rhy thmic e f f e c t s of a 1 idoca ine ..con-gener, t o c a i n i d e , 2-amino-2 ' ,6 ' p r op i onoxy l i d i de i n man". C l i n . Pharmacol. Ther. 19: 396-402 (1976). 34. Wink le , R. A . , F i t z g e r a l d , J . W. and M e f f i n , P. J . , B e l l , P. A . , Harapat, S. R. and Ha r r i s on , D. C , "An t i a r rhy thmic and pharmaco-k i n e t i c p r ope r t i e s of W-36095 in man". The Pharmaco log is t 1_7: 262 (1975) [Ab s t . ] 35. Woosley, R. L., McDev i t t , D. G. , Smi th , R. F., N i e s , A. S . , W i l k i n son , G. R. and Oates, J . A . , "An t i a r rhy thm i c and pharmacok inet ics of 2-am ino -2 ' , 6 1 p r op i onoxy l i d i d e HC1 (W-36095 - HC1, A s t r a ) i n man". C i r c u l a t i o n . 52: 11-75 (1975) [Ab s t . ] 36. Woosley, R. L., McDev i t t , D. G., Smith, R. F., N i e s , A. S . , W i l k i n son , G. R. and Oates, J . A . , "An t i a r rhy thmic and pharmacokinet ic p r ope r t i e s of 2-amino 2 ' - 6 ' p r op i onoxy l i d i de hydroch lo r i de (W-36095-Astra) i n man". The Pharmacolog is t V7: 262 (1975) [Ab s t . ] 37. Meyer, M. B., L a l k a , D., Duce, B. R., E l v i n , A. T . , "Pharmacok inet i cs o f W-36095 i n normal v o l un t e e r s " . C l i n . Pharmacol . Ther . 1_7: 240 (1975) [ A b s t r . ] - 220 -38. R ichey, D. P. and Bender, A. D., "Pharmacok inet ic consequences of a g i n g " . Annu. Rev. Pharmacol. T o x i c o l . ]]_'. 49-65 (1977). 39. Benet, L. Z. ( e d . ) , "The E f f e c t of d i sease s t a t e s on drug pharmaco-k i n e t i c s " . American Pharmaceut ica l A s s o c i a t i o n . Washington, 1976. 40. Woosley, R. L., McDev i t t , D. G. , N i e s , A. S . , Smi th , R. F., W i l k i n son , G. R. and Oates, J . A . , "Suppress ion of v e n t r i c u l a r e c t op i c d epo l a r i z a t i o n s by t o c a i n i d e " . C i r c u l a t i o n 56: 980-984 (1977). 41. M e f f i n , P. J . , Wink le , R. A . , B laschke, T. F., F i t z g e r a l d , J . and Ha r r i s on , D. C , "Response op t im i z a t i o n of drug dosage: An t i a r rhy thm i c s tud i e s w i t h t o c a i n i d e " . C l i n . Pharmacol. Ther. 22: 42-57 (1977). 42. B e r l i n - Wahlen, A . , Barkus, J . C , Keenaghan, J . B.; Lebeaux, M. and Tenthorey, P. A . , " E l im i n a t i o n o f N-(2-amino a c y l ) - 2 , 6 - x y l i d i n e s a f t e r in t ravenous i n f u s i o n i n dogs". A c t a . Pharm. Suec ica 1_4: 417-424 (1977). 43. Gudzinowicz, B. J . and Gudzinowicz, M. J . , " Ana l y s i s o f drugs and metabo l i t es by gas chromatography-mass spe c t r ome t r y " .Vo l . 1 and 2. Marcel Dekker I n c . , New York (1977). 44. D ixon, P. F., Gray, C. H., L im, C. K., and S t o l l , M. S. (eds . ) "High pressure l i q u i d chromatography i n c l i n i c a l chem i s t r y " . Academic P ress . London (1976). 45. Venkataramanan, R., Axe l son , J . E., " E l e c t r on capture de tec to r G.L.C. technique f o r e s t ima t i ng t o c a i n i d e i n b i o l o g i c a l f l u i d s " . J . Pharm. S c i . 67: 201-205 (1978). 46. M e f f i n , P. J . , Harapat, S. R., and Ha r r i s on , D. C , "H igh-pressure l i q u i d chromatographic a n a l y s i s of drugs i n b i o l o g i c a l f l u i d s I I : Determinat ion of an an t i a r rhy thm i c drug, t o c a i n i d e as i t s dansyl d e r i v a t i v e us ing f l uo rescence d e t e c t o r " . J . Pharm. S c i . 66_: 583-586 (1977). 47. Remmer, H., "Drugs as a c t i v a t o r s of drug enzymes". In B rod i e , B. B. and Erdos, , E. G. (eds. ) "Metabo l i c f a c t o r s c o n t r o l l i n g du ra t i on of drug a c t i o n " . Proceedings o f the F i r s t I n t e rna t i ona l Pharmacolog ica l Meet ing. V o l . 6. The Macmi l lan Company, N.Y. 1962, pp. 235-256. 48. Conney, A. H., "Environmental f a c t o r s i n f l u e n c i n g drug metabo l i sm". In La Du, B. N., Mandel, H. G. and Way, E. L., "Fundamentals of drug metabol ism and drug d i s p o s i t i o n " . The W i l l i ams and W i l k i n s Company, Ba l t imore (1971), pp. 253-278. 49. Remmer, H., " Induc t ion o f drug me tabo l i z i ng enzymes i n d i f f e r e n t animal s pe c i e s " . In Baker, S. B. DE. C..-Tripod, J . and Jacob, J . , "The problems of spec ies d i f f e r e n c e and s t a t i s t i c s i n t o x i c o l o g y " . Pro-ceedings o f the European So c i e t y f o r the Study of Drug T o x i c i t y , V o l . X I , Excerpta Medica, Amsterdam, 1970, pp. 14-18. - 221 -50. Conney, A. H., Dav ison, C , G a s t e l , R., and Burns, J . J . , "Adapt ive increase in drug metabo l i z i ng enzymes induced by phenobarb i ta l and o ther drugs" . J . Pharmacol. Exp. Ther. 130: 1-8 (1960). 51. V a l e r i n o , D. M., V e s s e l , E. S . , A u r o r i , K. C , and Johnson, A. 0 . , " E f f e c t o f va r i ous b a r b i t u r a t e s on hepat i c microsomal enzymes.. A comparat ive s tudy" . Drug Metab. D ispos. 2: 448-457 (1974). 52. Breckenr idge , A . , Orme, M. L., Dav ies , L., Tho rge i r s son , S. S. and Dav ies , D. S . , "Dose dependent enzyme i n d u c t i o n " . C l i n . Pharmacol. Ther. 14: 514-520 (1973). 53. Pe lkonen. 0 . , and K a r k i , N. J . , " E f f e c t of phys iochemica l and pharmaco-k i n e t i c p r ope r t i e s of b a r b i t u r a t e s on the i nduc t i on o f drug metabo l i sm". Chem. B i o l . I n t e r a c t . 7: 93-99 (1973). 54. Remmer, H., " Induc t i on of drug me tabo l i z i ng enzyme system i n the l i v e r " . Eur. J . C l i n . Pharmacol. 5_: 116-136 (1972). 55. Dutton, G. J . , Hanson, K. B., and B u r c h e l l , B., " E f f e c t o f pheno-b a r b i t a l and other compounds on i nduc t i on or maintenance of l i v e r u r id ined iphosphate g lucurony l t r ans f e r a se dur ing organ c u l t u r e " . Biochem. J . 120: 15 p. (1970). 56. Henderson, P. Th . , Phenobarb i ta l i nduc t i on o f UDP-glucuronyl t r a n s -fe rase i n i s o l a t e d r a t hepatocytes" . L i f e S c i . 1_0: 655-660 (1971 ) . 57. Tha l e r , M. M., Dal lman, P. R., and Goodman, J . , "Phenobarb i ta l induced changes i n NADPH-cytochrome e^reductase and smooth endoplasmic r e t i c u l um i n human. l i ve r " . J . P aed i a t r . 80: 302-310.(1972). 58. O r ren iu s , S . , E r i c s s o n , J . L. E. and E r n s t e r , L., "Phenobarb i ta l i n -duced syn thes i s of microsomal drug metabo l i z i ng enzymes and i t s r e l a -t i o n sh i p to p r o l i f e r a t i o n o f endoplasmic r e t i c u l u m " . J . C e l l . B i o l . 25: 627-639 (1965). 59. Burns, J . J . , Conney, A. H., and Kos te r , R., "S t imu l a t o ry e f f e c t of chron ic drug adm in i s t r a t i o n on drug metabo l i z i ng enzymes i n l i v e r microsomes".. Ann. NY Acad. S c i . 104: 881-893 (1963). 60. Fever, G., and Granda, V . , " An t agon i s t i c e f f e c t of f o r e i gn compounds on microsomal enzymes o f the l i v e r o f the r a t " . T o x i c o l . A p p l . Pharmacol. 16.: 626-637 (1970). 61. Ave l l aneda , M., " I n t e r f e r e n c i a de l o s b a r b i t u r i c o s en l a acc ion del tromexan". Medic ina (Buenes A i r e s ) 15_: 109-115 (1955) [ c i t e d by] Shand, D. G. , M i t c h e l l , J . R. and Oates, J . A . , "Pharmacok inet i c drug i n t e r a c t i o n s " . In G i l l e t t e , J . R. and M i t c h e l l , J . R. (eds . ) Handbook of Exper imenta l Pharmacology V o l . 28(3) . Sp r i n ge r -Ve r l a g , B e r l i n N.Y. 1975, pp. 272-314. 62. MacDonald, M. G . , Robinson, D. S . , S y l v e s t a , D., J a f f e , J . J . , "The e f f e c t s of phenoba rb i t a l , c h l o r a l beta ine and g l u t e th im ide - 222 -adm in i s t r a t i o n on wa r f a r i n plasma l e v e l s and hypoprothrombinemic responses i n man". C l i n . Pharmacol. Ther. 1TJ: 80-84 (1969). 63. Heinonen, J . , " In f l uence o f some drugs on t o x i c i t y and ra te of meta-bo l i sm of l i d o c a i n e and mepivacaine exper imental study on mice and r a t s " . Ann. Med. Exp. B i o l . Fenn. 44: sup 3, 1-43 (1966). 64. Ne t t e r , K. J . , "Drugs as i n h i b i t o r s of drug metabo l i sm". In B rod i e , B. B. and Erdos, E. G. ( e d . ) , "Metabo l i c f a c t o r s c o n t r o l l i n g du ra t i on of drug a c t i o n " . Proceedings of the F i r s t I n t e r na t i ona l Pharmacolo-g i c a l Meet ing. Vo l - 6 , The Macmi l lan Company (1962), pp. 213-233. 65. Kato, R, Ch i e sa ra , E., and V a s s a n e l l i , P., "Fur ther s tud ies on the i n h i b i t i o n and s t imu l a t i o n of microsomal drug metabo l i z i ng enzymes i n r a t l i v e r by va r i ous compounds". Biochem. Pharmacol. J_3: 69-83 (1964). 66. V e s s e l , E. S. and Passanan t i , G. T . , " I n h i b i t i o n of drug metabol ism i n man". Drug Metab. D ispos. ]_: 402-410 (1973). 67. Hansen, J . M., K r i s t en sen , M., Skovs ted , L., and Ch r i s t ensen , L. K., "Dicoumarol induced d ipheny lhydanto ih i n t o x i c a t i o n " . Lancet 2: 265-266 (1966). 68. K r i s t e n s en , M. and Hansen, J . M., " P o t e n t i a t i o n o f the to lbutamide e f f e c t by d i coumaro l " . D iabetes . 16^ : 211-214 (1967). 69. Ch r i s t ensen , L. K., Hansen, J . M., and K r i s t e n s e n , M., "Sulphaphenazole inudced hypoglyemic a t t a cks in to lbutamide t r ea ted d i a b e t i c s " . Lancet , 2: 1298-1301 (1963). 70'.. Huston, D. H., "Mechanisms of b i o t r an s f o rma t i o n " . In Hathway, D. E. ( sen io r r e p o r t e r ) . "Fore ign compound metabol ism i n mammals". Vo l -3 The Chemical S o c i e t y , Bu r l i ng t on House, London (1975), p. 495. 71. S t i t z e l , R. E., Stevens, J . T. and M c P h i l l i p s , J . J . , " E f f e c t s of environmental organophosphorous i n s e c t i c i d e s on drug metabo l i sm". Drug Metab. Rev. 1: 229-248 (1973). 72. Stevens, J . T . , S t i t z e l , R. E, and M c P h i l l i p s , J . J . , " E f f e c t s of a n t i c h o l i n e s t e r a s e i n s e c t i c i d e s on hepat i c microsomal matabo l i sm". J . Pharmacol. Exp. Ther. 181: 576-583 (1972). 73. Ash l ey , J . J . and Levy, G. , " I n h i b i t i o n o f d ipheny lhydanto in e l i m i n a -t i o n by i t s major me t abo l i t e " . Res. Commun. Chem. P a t h o l . Pharmacol. 4: 297-306 (1972). 74. Cook, L., Macko, E., F e l l o u s , E. J . , "The e f f e c t of g -d ie thy lamino-e thy l d i pheny l p ropy l aceta te , hyd roch lo r i de on the a c t i on o f a s e r i e s of b a r b i t u r a t e s and C. N. S. dep ressan t s " . J . Pharmacol. Exp. Ther. 112: 382-386 (1954). - 223 -75. Cooks, L., Nav i s , G. , and Fe l l ows , E. J . , "Enhancement of the a c t i o n of c e r t a i n a na l g e t i c drugs by 3 -d ie thy l aminoe thy ld ipheny lp ropy lace ta te hyd ro ch l o r i d e " . J . Pharmacol. Exp. Ther. 112: 473-479 (1954). 76. Axe l r od , J . , Re i chen tha l , J . , and B rod i e , B. B., "Mechanism of the p o t en t i a t i n g a c t i on of 3 -d i e thy l am inoe thy l d i pheny l p ropy l a ce ta t e " . J . Pharmacol. Exp. Ther. JJ_2: 49-54 (1954). 77. Cooper, J . , A xe l r od , J . , and B rod i e , B. B., " I n h i b i t o r y e f f e c t s o f 3 -d i e thy l aminoethy ld ipheny lpropy l acetate, on a v a r i e t y of drug metabo l i c pathways i n v i t r o " . J . Pharmacol. Exp. Ther. 112: 55-63 (1954). 78. Ikeda, M., Tanaka, S. and Katayama, T . , " I n h i b i t o r y e f f e c t o f SKF 525-A on aromat ic hyd roxy l a t i on and pheno l i c g l u c u r o n i d a t i o n " . Mol . Pharmacol. 4: 38-43 (1968). 79. Fouts, J . R. and B rod i e , B. B., " I n h i b i t i o n of drug metabo l i c path-ways by the p o t e n t i a t i n g agent, 2, 4, d ich loro-6-pheny l phenoxyethy l -d i e thy l am ide " . J . Pharmacol. Exp. Ther. 115: 68-73 (1955). 80. Anders, W., and Mannering, G. J . , " K i n e t i c s o f i n h i b i t i o n o f N-demethy lat ion of ethylmorphine by 2 -d i e thy l aminoethyl 2,2-d iphenyl -v a l e r a t e HC1 (SKF 525-A) and r e l a t e d compounds". Mo l . Pharmacol. 2: 319-327. (1966). 81 . Fouts, J . R- and B rod i e , B. B., "The enzymatic r educ t i on o f c h l o r -amphenicol p -n i t robenzo i c a c i d and other aromat ic n i t r o compounds i n mammals". J . Pharmacol. Exp. Ther. 119, 197-207 (1957). 82. Gaudette, L. E. and B rod i e , B. B. " Re l a t i o n sh i p between the l i p i d s o l u b i l i t y o f drugs and t h e i r o x i d a t i o n by l i v e r microsomes". Biochem.Pharmacol. 2: 89-96 (1959). 83. G i l l e t t e , J.R... and Kamm, J . J ., "The enzymatic fo rmat ion of s u l f o x i d e s : The ox i da t i on of ch lorpromazine and 4 ,4 'd iaminod ipheny l s u l f i d e by guinea p ig l i v e r microsomes". J . Pharmacol . Exp. Ther:.•>. 130: 262-267 ( I960) . 84. Ho l l unger , G. , "Some c h a r a c t e r i s t i c s o f an amide hyd ro l y s i ng micros somal enzyme before and a f t e r i t s s o l u b i l i z a t i o n " . A c t a . Phacmacol. U : 384-389 (1960). 85. Akerman, B. and Ross, S . , " S t e r e o s p e c i f i c i t y o f the enzymatic b i o t r ans f o rma t i on o f the enantiomers of P r i l o c a i n e " . A c t a . Pharmacol. T o x i c o l . 28: 445-453 (1970). 86. Levy, G . , "Dose dependent e f f e c t s i n pharmacok ine t i c s " . In Tedesch i , D. H. and Tedesch i , R. E. (eds . ) "Importance of fundamental p r i n c i p l e s i n drug e v a l u a t i o n " . Raven P ress , N.Y. 1968, pp. 141-172. - 224 -87. George, C. F., B l a c k w e l l , E. W., and Dav ies , D. S . , "Metabol ism o f I sop rena l i ne i n the i n t e s t i n e " . J . Pharm. Pharmacol. 26: 265-267 (1974). 88. Handbook of s i l y l a t i o n - P i e r ce Chemical Company, Rock ford , I l l i n o i s (1972), p. 19. 89. B l au , K. and K ing , S. K., " A c y l a t i o n " . In B l au , K., and K ing , G. S. (eds.) "Handbook of d e r i v a t i v e s f o r gas chromatography". Heyden, London (1977), pp. 104-151. 90. Wa l l e , T . , and Ehrsson, H., "Quan t i t a t i v e gas chromatographic de te r -minat ion of picogram q u a n t i t i e s of amino and a l c o h o l i c compounds by e l e c t r on capture d e t e c t i o n . Par t 1. P repara t i on and p r ope r t i e s of hep ta f l uorobutyry l d e r i v a t i v e s " . A c t a . Pharm. Suec ica 7_: 389-406 (1970). 91. Weeks, J . R. and Dav i s , J . D., "Chron ic int ravenous cannulas f o r r a t s " . J . App l . P h y s i o l . 19: 540-541.(1964). 92. B l au , K., and K ing , G. S . , " A c y l a t i o n " . In B l au , K. and K ing, G..S:..-( eds.) "Handbook o f d e r i v a t i v e s f o r gas'chromatography". .Heyden, London (1 977), p. 108. 93. i b i d . , p. 109. 94. i b i d . , p. 119. 95. i b i d . , pp."; 139-141. 96. P a i l e r , M., and Hubsch, W. J . , Mh. Chem. 97, 1541 (1966). Through, B l au , K., and K ing , G. S. " A c y l a t i o n " . In B l au , K., and K ing , G. S . , "Handbook of d e r i v a t i v e s f o r gas chromatography"-. .Heyden, .London (1977), p. 147. 97. Vander Molen, H. J . , Van der Maas, J . H., and Groen, D., " P repa ra t i on and p r ope r t i e s of s t e r o i d hep t a f l uo r obu t y r a t e s " . Eur . J . S t e r o i d s . 2: 119-138 (1967). 98. Poo le , C. F. and Morgan, E. D., "Anomolies i n the gas l i q u i d chromato-graphy o f c ho l e s t e r o l hep t a f l uo r obu t y r a t e " . J . Chromatogr. 90: 380-381 (1974). 99. Anggard, E., and S e d v a l l , G . , "Gas chromatography of catecholamine metabo l i t es us ing e l e c t r on caputre de tec t i on and mass spec t rometry" . Ana l . Chem.41_: 1250-1256 (1969). 100. W i l k , S . , .Git low,. S. E., F r a n k l i n , M. J . , and Ca r r , H. E., "A mic ro-q u a n t i t a t i v e technique f o r the de t e c t i on of some b i o l o g i c a l l y important amines by g a s - l i q u i d chromatography". C l i n . Chem. Acta J_0: 193-195 (1964). - 225 -101. H i r o i , M., and Kushinsky, S . , "Gas chromatographic a n a l y s i s o f • es t rogens" , Mikrochim. A c t a . (Wien) 1160-1164 (1969). 102. Van der Molen, H. J . , Groen, D., "Determinat ion of progesterone i n human pe r i phe ra l blood us ing gas l i q u i d chromatography w i th e l e c t r on capture d e t e c t i o n " . J . C l i n . Endoc r i no l . Metab: 25: 1625-1639 (1965). 103. Mat in , S. B., and Rowland, M., " E l e c t r on c a p t u r e ^ s e n s i t i v i t y comparison of va r i ous d e r i v a t i v e s of pr imary and"secondary amines". J . Pharm. S c i . 61_: 1235-1240 (1972). 104. Sevc i k , J . , "Detec tors i n gas chromatography". Journal of chromato-graphy L i b r a r y V o l . 4, E l s e v i e r S c i e n t i f i c Pub l i s h i ng Company, Amsterdam, 1976, p. 80. 105. Wentworth, W. E., Chen, E., " E l e c t r on attachment mechanisms -A n a l y t i c a l importance of temperature and concen t ra t i on v a r i a b l e s " . J . Gas Chromatogr. 5: 170-179 (1967). 106. Mandel, H. G. , "Pathways of drug b i o t r ans f o rma t i on : Biochemical con juga t i ons " . In La Du, B. N., Mandel, H. G. and Way, E. L. (eds.) "Fundamentals of drug metabol ism and drug d i s p o s i t i o n " . The W i l l i ams and W i l k i n s Company, Ba l t imore (1971), pp. 149-186. 107. Ch iou, W. L., Riegelman, S . , and Amberg, J . R., "Compl i ca t ions i n us ing r a b b i t s f o r the study of o ra l drug a b s o r p t i o n " . Chem. Pharm. B u l l . (Tokyo) 17: 2170-2173 (1969). 108. Longnecker, D. E., Guest d i s c u s s i o n . During 45th Congress of the I n t e rna t i ona l Anaesthes ia Research So c i e t y , March 14-17, 1971, Ba lHarbour, F l o r i d a . Anaesth. Ana l g . ' 5 0 : 665-666 (1971). 109. Upton, P. K. and Morgan, D. J . , "The e f f e c t of sampling techniques on some blood parameters i n the r a t " . Lab. Anim. 9: 85-91 (1975). 110. Barnes, C. D., and E l t h e r i n g t o n , L. G. , "Drug dosage i n l abo ra to ry an ima l s , a handbook". U n i v e r s i t y of C a l i f o r n i a P ress , Be rke l ey , (1973), p. 22. 111. Van Dyke, R. A. and R ikans , L. E. E f f e c t s of the v o l a t i l e anaesthe-t i c s on a n i l i n e hydroxy lase and aminopyrine demethylase. Biochem Pharmacol. 19: 1501-1502 (1970). 112. L inde , H. W. and Berman, M. L., "Nonspec i f i c s t imu l a t i o n o f drug me tabo l i z i ng enzymes by i n h a l a t i o n anaes the t i c agents" . Anaesth. Ana lg . 50: 656-665 (1971). 113. Wang, L., "Plasma volume, c e l l volume, t o t a l b lood volume and F -|-j f a c t o r i n normal and sp lenectomized sherman r a t s " . Am. J . P h y s i o l . 1_96: 187-192 (1959). 114. G i b a l d i , M.,and P e r r i e r , D., "Pharmacok ine t i c s " . Marcel .Dekker (1976) pp. 68-69. - 226 -115. Chau, N. P., "Area-dose r e l a t i o n s h i p s i n non - l i n ea r models". J . Pharmacokinet. Biopharm. 4: 537-551 (1976). 116. F r a n k l i n , C. S. and Kyegombe, D. B., "The d i s t r i b u t i o n and i nduc t i on o f some drug metabo l i z i ng enzymes i n man". B r . J . Pharmacol . 47 (3 ) : 616 P (1973). 117. Kruger-Thiemer, E., "Non- l i nea r dose-concent ra t ion r e l a t i o n s h i p s " . Farmaco. ed. S c i . 28: 718-756 (1968). 118. Kruger-Thiemer, E., D i l l e r , W. and Bunger, P., "Pharmacok inet i c model regard ing p r o t e i n b ind ing of d rugs" . An t im i c rob . Agent. Chemotherap. 1_: 183-191 (1965). 119. McNamara, P. J . , Levy, G. and Gi lba ld i . , , M., " E f f e c t o f plasma p ro t e i n and t i s s u e b ind ing on the t ime x eourse of drug concen t ra t i on in p lasma". (Submitted f o r p u b l i c a t i o n ) . 120. Baggot, J . D. and Dav i s , L. E., "Comparative study of plasma p r o t e i n b ind ing o f d i pheny lhydan to in " . Comp. Gen. Pharmacol. 4: 399-404 (1973). 121. Wagner, J . , " P h a r m a c o k i n e t i c s - H a l f - l i f e and volume o f d i s t r i b u t i o n " . Drug I n t . C l i n . Pharm. 2: 126-131 (1968). 122. Disa'nto, A. R., "A new non - l i n ea r pharmacokinet ic model w i th s p e c i f i c a p p l i c a t i o n to methylene b lue" - a d i s s e r t a t i o n submitted i n p a r t i a l f u l f i l l m e n t o f the requirement f o r the degree of Doctor of Ph i losophy (pharmacy) U n i v e r s i t y of Mich igan (1971). 123. B i s c h o f f , K. B., Dedr i ck , R. L., Zaharko, D. S. and Longs t r e th , J . A. "Methotrexate pharmacok ine t i c s " . J . Pharm. S c i . 60: 1128-1133 (1971). 124. Kaye, C. M., K i dd i e , M. A. and Turner , P., " Va r i a b l e pharmacok inet ics of m e x i l e t i n e " . Pos tg rad . Med. J . 53 (Supp): 56-58 (1977). 125. Lev ine , W. G. , " B i l i a r y e x c r e t i on of drugs and other x e n o b i o t i c s " . In George, R., Okun, R., and Cho, A. K. (eds ) . Annu. Rev. Pharmacol. T o x i c o l . , pp. 81-96 (1978), Annual Reviews Inc . C a l i f o r n i a . 126. Levy, G. , Voge l , A. W. and Amsel , L. P., "Capac i t y l i m i t e d sa l i c y ! u r •; r a t e format ion dur ing prolonged adm in i s t r a t i o n of a s p i r i n to hea l thy human sub j e c t s " . J . Pharm. S c i . 58: 503-504 (1969). 127. Drucker, M. M., Blondheim, S. H., and W i s l i c k i , L., " Fac to r s a f f e c t i n g a c e t y l a t i o n i n v i vo o f para-amino-benzoic a c i d by human s u b j e c t s " . C l i n . S c i . 27: 133-141 (1964). 128. Levy, G. , "Pharmacok inet i cs of s a l i c y l a t e e l i m i n a t i o n i n man". J . Pharm. S c i . 54: 959^967 (1965). - 227 -129. Boxer, G. E., J e l i n e k , V. C , Tompseth, R., Dubois, R. and Ed i son , A. 0 . , "Streptomyc in i n the b lood: chemical de terminat ion a f t e r s i n g l e and repeated in t ramuscu la r i n j e c t i o n s " . J . Pharmacol. Exp. Therap. 92: 226-275 (1948). 130. Levy, G. , Tsuch iya , T. and Amsel , L. P., " L im i t ed capac i t y f o r s a l i c y l p h eno l i c g lucuron ide format ion and i t s e f f e c t on the k i n e t i c s o f s a l i c y l a t e e l i m i n a t i o n in man". C l i n . Pharmacol. Ther. 1_3: 258-268 (1972). 131. Weiner, M., Shap i r o , S . , A xe l r od , J . , Copper, J . R., and B rod ie , B. B. "The phy s i o l o g i c a l d i s p o s i t i o n of d icumarol i n man". J . Pharmacol. Exp. Therap. 99 -409-420 (1950). 132. B rod i e , B. B., Weiner, M., Burns, J . J . , Simon, G. and Y a l , E. K., "The phy s i o l o g i c a l d i s p o s i t i o n of e thy l b iscoumacetate i n man and a method f o r i t s e s t ima t i on i n b i o l o g i c a l m a t e r i a l s " . J . Pharmacol. Exp. Therap. J06 : 453-'463 (1952). 133. Dayton, P. G. , Yu, T; F., Chen, N., Berger , L., West, L. A . , and Gutman, A. B., "The p h y s i o l o g i c a l d i s p o s i t i o n of probenec id i n c l u d i n g renal c learance i n man s tud ied by an improved method f o r i t s e s t ima-t i o n i n b i o l o g i c a l m a t e r i a l " . J . Pharmacol. Exp. Therap. 140: 278-286 (1963). 134. Dayton, P. G. , C u c i n e l l , S. A . , Weiss, M., and P e r e l , J . M., "Dose dependence of drug plasma l e ve l d e c l i n e in dogs". J.• Pharmacol. Exp. Therv . 158: 305-310' (1967). 135. Jahnchen, E. and Levy, G. , " I n h i b i t i o n o f phenylbutazone e l i m i n a t i o n by i t s metabo l i t e oxyphenbutazone". P roc . Soc. Exp. B i o l . Med. 141: 963-965 (1972). 136. Ash l ey , J . J . and Levy, G. , " I n h i b i t i o n of d ipheny lhydanto in e l i m i n a t i o n by i t s major me t abo l i t e " . Res. Commun. Chem. P a t ho l . Pharmacol. 4: 297-306 (1972). 137. Borondy, P., Chang, T . , and G lazko , A. J . , " I n h i b i t i o n of d iphenyl hydantoin hyd roxy l a t i on by 5-p-hydroxyl=5-phenylhydantoin. ; (p-HPPH)". Fed. Proc. 31_: 582 (1972) [Ab s t . ] 138. Smith, R. L. and W i l l i ams , R. T . , " H i s t o r y and the d i s cove ry of the con jugat ion mechanisms". In Fishman, W. H. (ed.) "Metabol ic- con-juga te and metabo l i c h y d r o l y s i s " . V o l . 1, Academic P re s s , New York (1970), pp. 1-19. 139. Axe l r od , J . E., Inscoe, J . K., and Tomkins, G. M., "Enzymat ic syn-t he s i s of N-g lucosy lu ron i c a c i d con juga tes " . J . B i o l . Chem. 232: 835-841 (1958). - 228 -140. Marsh, C. A . , "Chemistry o f D-g lucuronic a c i d and i t s g l y c o s i d e s " . In Dutton, G. J . (ed.) "G lucuron i c a c i d f r ee and combined". Academic Press (1966), pp. 3-136. 141. Levvy, G. A. and Marsh, C. A . , "The p repara t i on and fp rope r t i e s of 3 -g lucuron idase i n h i b i t i o n by sugar a c i d s and l a c t one s " . Biochem. J . 52: 464-472 (1952). 142. Levvy, G. A . , and Marsh, C. A . , "The p repa ra t i on and p rope r t i e s of 3- g lucuron idase s p e c i f i c i t y of 3 -g lucu ron idase" . Biochem. J . 52; 690-694 (1952). 143. Wakabayashi, M., " 3 -g lucuron idases i n metabo l i c h y d r o l y s i s " . In Fishman, W. H., "Metabo l i c con jugat ion and metabo l i c h y d r o l y s i s " , V o l . I I , Academic P ress , New York (1970), pp. 520-603. 144. R i cha rds , L. G. , C a s t l e , M. C. and Lage, G. L. "GIucuron ida t i on o f d i g i t o x i n and i t s d e r i v a t i v e s by r a t and r a b b i t l i v e r homogenates". Drug Metab. Dispos. 5_: 469-473 (1977). 145. D i rnhuber, P. and S c h u l t z , F., "The i somer i c t r a n s i t i o n of urea i n t o ammonium cyanate i n aqueous s o l u t i o n s " . Biochem. J . 42: 628-632 (1948). 146. S t a r k , G. R., S t e i n , W. H., Moore, S . , "React ions of the cyanate present i n aqueous urea w i th amino ac i d s and p r o t e i n s " . J . B i o l . Chem. 235: 3177-3181 (1960). 147. Evans, M. E., Walker, S. R., B r i t t a i n , R. T. and Pa te rson , J . W., "The metabol ism of salbutamol i n man". Xenob i o t i c a . 3; 113-120 (1973). 148. Rub in, A . , Warr i ck , P., Wolen, R. L., Chern i sh , M. S . , R i d o l f o , A. S. and Gruber, C.. M. J r . : , \ " Phy s i o l o g i c a l d i s p o s i t i o n of feno-profen i n man. I I I . Metabol ism and p r o t e i n b ind ing of f enopro fen" . J . Pharmacol . Exp. Therap. ] 8 3 : 449-457 (1972). 14 149. Cramma, J . L., S c o t t , B. and Ro l f e , B., "Metabol ism of C- imipramine: II u r i n a r y metabo l i t es i n man". Psychopharmacologie. 1_5: 207-225 (1969). 150. Ne lson, S. D., Breck, G. D. and Trager , W. F. " In v i vo metabo l i c con-densat ions. Formation o f N^ethy l -2 -methyT-N 3 - (2 ,6 dimethyl phenyl) 4- im idazo l i d i none from the r e a c t i o n of a metabo l i t e of a l coho l w i th a metabo l i t e o f l i d o c a i n e " . J . Med. Chem. U: 1106-1112 (1973). 151. Morgan, D. J . , Smyth, M- P-> Thomas, J . and V ine , J . , " C y c l i c metabo l i t es of e t i d o ca i ne in humans". Xenob io t i ca 7_: 365-375 (1977). 152. Ma r i e r , J . R. and Rose, D., "Determinat ion of cyanate, and a study of i t s accumulat ion in aqueous s o l u t i o n s of u r ea " . Ana l , biochem. 7: 304-314 (1964). - 229 -1.53. Nye, J . F. and M i t che l l , H. K., Synthesis of orot ic acid from aspart ic ac id . J . Am. Chem. Soc. 69: 1382-1384 (1947). 154. Jones, M. E., "Par t i a l reactions of carbamyl-P synthetase:. A review and enquiry into the ro le of carbamate". In G r i s o l i a , S., Baguena, R., and Mayor, F. (eds.) "The urea cyc le" , John Wiley and Sons, N.Y. (1976), pp. 107-122. 155. Faurholt, C. "Uber die Prozess,, NH2C00NH4 + H 2 Q ^ (NH 4 ) 2 C0 3 "und,, C02+ H20 H 2C0 3". Z." Anorg. A l l gem. Chem. 120: 85-102 (1921 ). 156. Stark, G. R., and Smyth, D. G., "The use of cyanate for the deter-mination of NhL-terminal residues in prote ins". J . B i o l . Chem. 238: 214-225 (T963). 157. Oberholzer, V. G., and Palmer T., "Increased excretion of N-carbamoyl compounds'in patients with urea cycle defects". C l i n . Chim.; Acta. 68: 73-78 (1976). 158. Carreras, J . , Chabas, A., and Diedr ich, D., "Physio logical and C l i n i c a l Implications of Protein Carbamylation". In G r i s o l i a , S., Baguena, R. and Mayor, F., (eds.) "The urea .cycle", John Wiley and Sons, New York (1976), pp. 501-548. 159. Gros, G., Forster, R. E. and L i n , L., "The carbamate reaction of g lycy lg lyc ine plasma and t issue extracts evaluated by a pH stopped flow apparatus". J . B i o l . Chem. 25J_: 4398-4407 (1976). 160. Hansten, P. D., "Interact ion between anticonvulsant drugs: Primidone, diphenylhydantoin ; and phenobarbital". Northwest Med. J . 8: 17-23 (1974). 161. P iafsky, K. M., S i t a r , D. S. and Og i l v ie , R. I . , "E f fect of pheno-barbi ta l on the d ispos i t ion of intravenous theophyl l ine". C l i n . Pharmacol. Ther. 22: 336-339 (1977). 162. Landay, R. A., Gonzalez, M. A., and Taylor, J . C., "Ef fect of pheno-barb i ta l on theophyll ine d i spos i t i on" . J . A l l e r g . C l i n . Immunol. 62: 27-29 (1978). 163. Breckenridge, A., "C l i n i ca l impl icat ions of enzyme induct ion". In Parke, D. V., "Enzyme induct ion". Plenum Press. London (1975) pp. 273-302. 164. Lechner, M. C , and Gomes, F. P., "Enzyme.induction by phenobarbital and l i v e r microsomal RNase a c t i v i t i e s in mice, hamster, and guinea p igs". Gen. Pharmacol 6: 127-132 (1975). 165. Ohnhaus, E. E., Thorgiersson, S. S., Davies, D. S. and Breckenridge, „ ' A.,"Changes in l i v e r blood flow during enzyme induction". Biochem. Pharmacol. 20: 2561-2570 (1971 ).. - 230 -166. Branch, R. A., Shand, D. G., Wilkinson, G. R. and Nies, A. S., "Increased clearance of ant ipyr ine and d-propranolol a f te r pheno-barbita l treatment in the monkey". J . C l i n . Invest. J53: 1101-1107 (1974). 167. Klassen, C. D., " B i l i a r y flow a f te r microsomal enzyme induct ion". J . Pharmacol. Exp. Ther, JJ58: 218-223 (1969). 168. Klassen, C. D.,"Effect of phenobarbital on the plasma disappearance and b i l i a r y excretion of drugs, in rats" . J . Pharmacol. Exp. Ther. 175: 289-300 (1970). 169. Reyes, H., Lev i , A. J . , Gatmaitan, Z., and Ar ian, I. M., "Organic anion-binding protein in rat l i v e r : Drug induction and i t s physio-log ic consequences". Proc. Nat l . Acad. S c i . USA 64: 168-170 (1969). 170. Kutt, H., "Biochemical and genetic factors regulat ing d i l an t i n meta-bolism in man". Ann. NY Acad. S c i . 179: 704-722 (1971). 171. Breyer, U., Junginger, W. and Vil lumsen, D., "Phenobarbital induced prolongation of h a l f - l i f e and a l te ra t ion of d i s t r i bu t i on of a phenothiazine drug metabolite in the r a t " . Biochem. Pharmacol 25: 2623-2630 (1976). 172. Heinonen, J . , Takki , S. and Jarho, L., "Plasma l idocaine leve ls in patients treated with potential inducers of microsomal enzymes". Acta. Anaesthesiol. Scand. J4_: 89-95 (1970). 173. Remmer, H., and Siegert , M., "Kumulation and El iminat ion von Pheno-ba rb i t a l " , Nauyn-Schmiedebergs. Arch. Exp. Path. Pharmak. 243: 479-494 (1972). 174. Mant i l l a -P la ta , B. and Harbison, R. D., "Ef fects of phenobarbital and SKF 525-A pretreatment, sex, l i v e r in jury and vehic le on A 9 -Tetrahydro-cannabinol t o x i c i t y " . Tox ico l . Appl. Pharmacol. 27: 123-130 (1974). 175. F i t zgera ld , T. J . , "Phenobarbital and SKF 525-A on v inb last ine and v i n c r i s t i ne t ox i c i t y in mice". Experientia 32: 619-620 (1976). 176. Goldstein, A., Aronow, L., and Kalman, S. M., "Pr inc ip les of drug act ion: The basis of pharmacology", John Wiley and Sons, New York (1974), p. 268. 177. Bus, J . S., "Ef fect of phenobarbital and SKF 525-A on the t ox i c i t y and metabolism of cyclophosphamide in new born mice". J . Pharmacol. Exp. Ther. 18_4: 749-756 (1973). 178. Goldste in, A., Aronow, L. and Kalman, S.M. (ed), "Pr inc ip les of drug ac t ion" , Harper and Row Publ ishers, New York, p. 250 (1968). - 231 -179. Bettschart, A., Be l l a , D., and Bovet, D., "Potenzierung der cur-areahnlichen Eigenschaften Von T815'..I.S.durch SKF 525-A". Helv. Phys io l . Acta. 14: C58-C59 (1956). Cited in Netter, K. J . , "Drugs as inh ib i to rs of drug metabolism". In Brodie, B. B. and Erdos E. G. (eds.) "Metabolic factors cont ro l l i ng duration of drug ac t ion" . Proceedings of the F i r s t International Pharmacological Meeting Vo l . 6. The Macmillan Company, New York.(1962), pp. 213-233. 180. Netter, K. J . , "Die Hemmung der Procainhydrolyse durch die Diathylaminoathanol-ester der Diphenylpropylessigsaure (SKF 525-A) und der Di phenyl essigaure ( t rasent ih ). Naunyn-Schmiedeberg' s Arch. Exp. Pathol. Pharm. 235: 498-512 (1959). 181. Jenner, S., and Netter, K. J . , "On the i nh ib i t i on of microsomal drug metabolism by SKF 525-^A". Biochem. Pharmacol. 21_: 1921-1927(1972). 182. Belpaire, F. M. 'Bogaert, M.G., "Metabolism, of papaverine". I l l Ef fects of phenobarbital, 3-methyl cholanthrene and SKF 525-A pretreatment in vivo and in v i t r o . Xenobiotica 5: 431-438 (1975). 183. Levine, W. G., "Effect of SKF 525-A on b i l i a r y funct ion. Influence of body temperature". L i f e . S c i . 9: 437-442 (1970). 184. Marchand, C. and Nad'eau:,D., "Ef fect of 2-diethylaminoethyl-2 ? 2-diphenylvalerate hydrochloride (SKF 525-A) on sulphacetamide d i s t r i bu t i on and excretion in r a t s " . Br. J . Pharmacol. 47: 69-76 (1973). 185. Hakim, R., and Fujimoto, J. M., " Inh ib i t ion of renal tubular transport of morphine by 3-diethyl aminoethyldiphenylpropTyacetate in the chicken". Biochem. Pharmacol. 20: 2647-2662 (1971). 186. Marchand, C. and Brodeur, J.,"Change in co l l o i da l gold clearance in rats treated with SKF 525-A". Rev. Canad. B i o l . 29: 293-298 (1970). 187. Lautt, W. W. et a l . , "The e f fec t of SKF 525-A and of a l tered hepatic blood flow on l idocaine clearance in cat" . Can. J . Phys io l . Pharmacol. 55: 7-12 (1977). 188. Akerman, B. and Ross, S., "Stereospec i f i c i ty of the enzymatic b io-transformation of the enantiomers of P r i l oca ine" . Acta. Pharmacol. Tox ico l . 28: 445-453 (1970). 189. Cook. L., Toner, J . J . and Fellows, E. J.,"The e f fec t of 3 -d ie thy l -aminoethyldiphenylpropyl acetate hydrochloride (SKF 525-A) on hexobarbita l" . J . Pharmacol. Exp. Ther, rn_: 131-141 (1954). 190. Singh v i , Si M., Conway, W. D., G iba ld i , M., and Shaar, S., "Ef fect of co-administered sal icylamide on terbutal ine metabolism in r a t s " . J . Pharm. S c i . 66: 1488-1489 (1977). 191. The Merck Index, Merck and Co. Inc. 1968, p. 930. - 232 -APPENDIX A Identity and Pur i ty of the Materials Tocainide Hydrochloride Melting Point 247°C [246°-247°] pKa 7.81 a Molecular Weight 228.5 Tocainide Base Melting Point 56.1°C Molecular Weight 192 Mass Spectrum Mass/Charge 44 121 192 106 120 77 91 Relative Intensity 100 92.9 65.9 39.1 36.8 23.4 15.2 a Astra Pharmaceutical Products, Inc. , Framingham, Massachusetts. - 233 -Glycine Xy l id ide Melting Point Molecular Weight Mass Spectrum Mass/charge 30 Relative in tens i ty 100 : a-Bromonaphthalene .Molecular Weight Mass Spectrum Mass/charge 206 Relative in tens i ty 100 178 148 121 178 106 15.28 15.21 11.52 11.05 208 208 127 63 103 96.3 96.3 28.5 14.8 - 234 Phenobarbital Sodium Melting Point 265-270 Molecular Weight 232.2 Mass Spectrum Mass/charge 204 Relative in tens i ty 57.8 melts with decomposition 117 141 77 156 146 29.3 26.7 , 19.8 . 13.8 12.9 SKF 525-A Melting Point 121.6°C Molecular Weight 353 Mass Spectrum Mass/charge 86 99 167 44 209 152 Relative in tens i ty 100 16.6 6.1 1.8 0.9 1.0 - 235 -Salicylamide Melting Point Molecular Weight Mass Spectrum Mass/charge Re lat ive/ intens i ty .140.4° (140°)a 137 120 137 92 65 39 100 66.1 36.7 13.1 10.2 a The Merck Index, 9th ed i t i on . - 236 -APPENDIX B NMR AND IR SPECTRA OF TOCAINIDE 3000 2000 " 1600 WAVELENGTH IN NANOMETERS. IR SPECTRUM OF TOCAINIDE. i r (KBr) 3280 cm 1 ( N l l 2 ) , 2800- 3000 cm 16^0 , 1530 cm" 1 ( amide I , amide I - 239 -APPENDIX C Evidence for the involvement of a metabolic component in the non-l inear e l iminat ion k inet ics of tocainide. As described in an ea r l i e r sect ion, 3 , the d ispos i t ion rate constant i s a function of the el iminat ion and d i s t r i bu t i on rate constants. Changes in the el iminat ion half l i f e of a compound due to a l terat ions in the value of 3 can, therefore,resu l t from a change in the d i s t r i bu t i ona l or e l iminat ion parameters or both. The fol lowing approach was used to separate the el iminat ion component from the overal l d ispos i t ion process and to study the ef fect of dose, on the e l iminat ion process, alone. With compounds exh ib i t ing two compartments k inet ic cha rac te r i s t i c s , the tota l body clearance (Vg3) i s given by the equation (1). 5 r C- dt J. where = apparent volume of d i s t r i bu t i on of the drug, 3 = d ispos i t ion rate constant XQ = the dose administered 0 0 * . rC dt = area under the plasma concentration versus time 0 J curve The tota l body clearance i s , in fac t , equal to the drug clearance from the central compartment when the e l iminat ion process occurs only from the central compartment. Total body clearance = V g 3 = V £ K E (2) where Vg and 3 are as defined-, ea r l i e r - 240 -V = volume of the central compartment c = el iminat ion rate constant from central compartment With compounds el iminated both by metabolism and renal excret ion, the renal clearance (Q) can be defined as Q = ke . vc (3) where k„ = renal excretion rate constant e Dividing equation (3) by equation (2) Renal clearance k V _ R /«\ Total body clearance p — T T — * ' E V c which i s equal to _e and hence a constant under l inear k ine t i c s . KE The el iminat ion rate constant i s composed of the excretion rate constant and the appropriate metabolic formation rate constants. KE = k e + kml + km2 kmi <5> A change in the value of "R" at d i f fe rent dose leve ls w i l l indicate a change in k or k , ,k 0 k ., or both. I f , however, the excretion 3 e ml md mi • rate constant k g remains constant, any change in the ra t i o "R" w i l l indicate a change in k^ , k^ or k^ .^ Such a s i tuat ion could occur under saturation k ine t i c s , enzyme induction or enzyme i nh i b i t i on . Table XXI summarizes the ra t io "R" obtained in the present study at d i f fe rent dose leve ls of tocainide. The renal clearance of tocainide was not s i gn i f i c an t l y d i f fe rent at dose leve ls of 5, 10, 15 and 20 mg/kg. Assuming that V c did not change with dose, then the value k g i s a constant. That i s , the renal excretion rate constant has not changed with higher dose. Since, the calculated value - 241 -of R increases at dose level of 20 mg/kg by nearly 100% th i s suggests a reduction in the value of K^ . This can be interpreted in terms of reduction in the contr ibut ion of the metabolic component to the tota l e l iminat ion of the drug as might be expected under the presence of saturable metabolic pathway.