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Pharmacokinetics, tissue distribution, and pharmacodynamics of valproic acid and its unsaturated metabolites… Lee, Ronald Duane 1991

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PHARMACOKINETICS, TISSUE DISTRIBUTION, AND PHARMACODYNAMICS OF VALPROIC ACID AND ITS UNSATURATED METABOLITES IN RATS RONALD DUANE LEE B . S c . ( P h a r m . ) , U n i v e r s i t y of B r i t i s h Columbia, Canada, 1982 M . S c , U n i v e r s i t y of B r i t i s h Columbia, Canada, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Faculty of Pharmaceutical Sciences D i v i s i o n of Pharmaceutical Chemistry We accept t h i s t h e s i s as conforming to the required standard U n i v e r s i t y of B r i t i s h Columbia A p r i l 1991 © R o n a l d Duane Lee, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Faculty of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada Date A p r i l 17, 1991  DE-6 (2/88) 1 1 ABSTRACT V a l p r o i c a c i d (VPA), an a n t i e p i l e p t i c drug, possesses a delay i n maximum pharmacological response upon i n i t i a l drug a d m i n i s t r a t i o n , and a prolonged duration of a c t i v i t y f o l l o w i n g d i s c o n t i n u a t i o n of the drug. Metabolites of VPA are thought to be involved as evidence from previous studies i n mice demonstrated that (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA, major products of VPA metabolism i n serum, exerted some degree of anticonvulsant a c t i v i t y against pentylenetetrazole (PTZ)-induced s e i z u r e s . Also associated with VPA therapy i s a f a t a l i d i o s y n c r a t i c h e p a t o t o x i c i t y p o s s i b l y i n v o l v i n g two m e t a b o l i t e s , 4-ene VPA and (E)-2 , 4 - d i e n e VPA. Preliminary t i s s u e d i s t r i b u t i o n studies had suggested that (E)-2-ene VPA may not be as hepatotoxic as VPA based s o l e l y on (E)-2-ene VPA concentrations i n l i v e r . The main o b j e c t i v e s of t h i s study were to i n v e s t i g a t e the k i n e t i c and metabolic p r o f i l e s , d i s p o s i t i o n , and anticonvulsant a c t i v i t y of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s . Results of these experiments were intended to provide i n s i g h t i n t o the p o s s i b l e c o n t r i b u t i o n s of these metabolites towards VPA a c t i v i t y or t o x i c i t y . Synthesis of (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA was accomplished by the r e g i o s p e c i f i c a d d i t i o n of propionaldehyde to an e s t e r e n o l a t e , followed by n u c l e o p h i l i c e l i m i n a t i o n of the mesylate e s t e r with l , 8 - d i a z a b i c y c l o [ 5 . 4 . 0 ] u n d e c - 7 - e n e or potassium hydride. The synthesis provided good y i e l d s and was s t e r e o s e l e c t i v e . The isomeric p u r i t y of the synthesized compounds was found to be 95 - 97% based on nuclear magnetic resonance and gas chromatographyc-mass spectrometric d a t a . The assay of VPA and i t s metabolites i n r a t plasma and t i s s u e homogenate e x t r a c t s was achieved by negative ion chemical i o n i z a t i o n gas chromatography-mass spectrometry. This method proved to be s e l e c t i v e , s e n s i t i v e , r e p r o d u c i b l e , and amenable to automation. In order to compare the d i s p o s i t i o n and pharmacokinetics of VPA and i t s analogues, VPA was administered i n t r a p e r i t o n e a l l y to r a t s and the k i n e t i c p r o f i l e s i n plasma, l i v e r , h e a r t , l u n g s , and nine brain regions were determined. S e l e c t i v e binding of VPA to l i v e r was observed with the l i v e r / p l a s m a r a t i o at 10 hours a f t e r dosing being 4 . 6 . VPA d i d not p e r s i s t i n brain and the d i s t r i b u t i o n i n brain t i s s u e appeared uniform. Metabolites of VPA a l s o were not retained i n b r a i n . A most i n t e r e s t i n g observation was the absence of ( E , E ) - 2 , 3 ' - d i e n e VPA i n brain w h i l e a minor plasma m e t a b o l i t e , ( E , Z ) - 2 , 3 ' - d i e n e VPA, was the only d e t e c t a b l e d i e n e . A s t e r e o s e l e c t i v e a c t i v e transport mechanism could account f o r t h i s unusual r e s u l t . Present i n plasma but not detected i n l i v e r was ( E ) - 2 , 4 - d i e n e VPA, the hepatotoxic metabolite of VPA. It was proposed that the diene may be c o v a l e n t l y bound to l i v e r t i s s u e . Following s i n g l e dose a d m i n i s t r a t i o n to r a t s , (E)-2-ene VPA appeared to p e r s i s t i n a l l t i s s u e s assayed f o l l o w i n g an i n i t i a l d e c l i n e phase. The prolonged terminal e l i m i n a t i o n phase may be a t t r i b u t e d to the extensive plasma p r o t e i n binding of (E)-2-ene VPA (>99%). No s e l e c t i v e binding of (E)-2-ene VPA i n brain was observed. Brain/plasma r a t i o s at 10 hours a f t e r dosing d i d not exceed 0 . 0 3 . Metabolites of (E)-2-ene VPA were mainly products of /}-oxidation and r e d u c t i o n . Both hepatotoxic metabolites were observed i n plasma with concentrations of 4-ene VPA i n l i v e r higher than normally seen f o l l o w i n g VPA a d m i n i s t r a t i o n . Questions a r i s e regarding the p o t e n t i a l h e p a t o t o x i c i t y of (E)-2-ene VPA. A f t e r s i n g l e dose a d m i n i s t r a t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s , clearance of the diene was r a p i d compared to that of VPA or (E)-2-ene VPA. S e l e c t i v e binding of the diene was observed i n the s u p e r i o r and i n f e r i o r c o l l i c u l u s and s u b s t a n t i a n i g r a but the concentrations were too low to be considered c l i n i c a l l y s i g n i f i c a n t . Reduction of ( E , E ) - 2 , 3 ' -diene VPA appeared to be the main route of metabolism. 4-Ene VPA and ( E ) - 2 , 4 - d i e n e VPA were not detected i n plasma or t i s s u e s suggesting ( E , E ) - 2 , 3 ' - d i e n e VPA may have a lower p o t e n t i a l f o r l i v e r t o x i c i t y . The anticonvulsant a c t i v i t i e s of VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA were compared i n r a t s by the PTZ-induced seizure t e s t . Based on ED50 v a l u e s , the anticonvulsant potencies of VPA and (E)-2-ene VPA were comparable and s i g n i f i c a n t l y greater than ( E , E ) - 2 , 3 ' - d i e n e VPA. The d e t e c t i o n of ( E , Z ) - 2 , 3 ' - d i e n e VPA i n brain f o l l o w i n g VPA a d m i n i s t r a t i o n l e d to the t e s t i n g of t h i s diene isomer. The potency of the (E ,Z)-isomer was found to be equivalent to VPA and (E)-2-ene VPA. Sedation was a severe side e f f e c t of (E)-2-ene VPA and the (E ,E)-2 , 3 ' - d i e n e VPA was s t e r e o s e l e c t i v e l y unique i n causing s k e l e t a l muscle r i g i d i t y . Sedation was minimal and muscle r i g i d i t y was not a property of the (E ,Z)-isomer over the dose range s t u d i e d . Based on the r e s u l t s of these s t u d i e s , i t can be concluded that n e i t h e r (E)-2-ene VPA nor ( E , E ) - 2 , 3 ' - d i e n e VPA i s r e s p o n s i b l e f o r the pharmacodynamic e f f e c t s of VPA. From the metabolism of ( E , E ) - 2 , 3 ' - d i e n e VPA and the r e s u l t s of anticonvulsant t e s t i n g , i t was proposed that ( E , Z ) - 2 , 3 ' - d i e n e VPA may have p o t e n t i a l as a r e l a t i v e l y safe and useful anticonvulsant drug. V TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Figures x i i L i s t of Tables x v i i i L i s t of Schemes xx Symbols and Abbreviations xxi Acknowledgements xxiv 1. INTRODUCTION 1 1.1 V a l p r o i c A c i d 1 1.1.1 Overview 1 1.1 .2 Adverse Reactions 3 1 . 1 . 2 . 1 Type A Adverse E f f e c t s of VPA 6 1 . 1 . 2 . 2 Type B Adverse E f f e c t s of VPA H e p a t o t o x i c i t y 7 1 . 1 . 2 . 3 Teratogenic E f f e c t of VPA 11 1 .1 .3 Metabolism 11 1.1 .4 Mechanism of A c t i o n 16 1 .1 .4 .1 The GABA System 16 1 . 1 . 4 . 2 Increase of Brain GABA 17 1 . 1 . 4 . 3 P o t e n t i a t i o n of Postsynaptic GABA 18 1 . 1 . 4 . 4 D i r e c t Membrane E f f e c t 19 1 .1 .5 Pharmacokinetics 19 1.1 .6 Pharmacodynamics 21 vi 1.1.7 Anticonvulsant A c t i v i t y of VPA Metabolites 22 1.2 Anticonvulsant Evaluation 24 1.2.1 E l e c t r i c a l l y Induced Seizures 24 1 . 2 . 2 Chemically Induced Convulsions 25 1.3 Objectives 27 1.3.1 S p e c i f i c Aims 27 2. EXPERIMENTAL 29 2.1 Supplies 29 2.1.1 Chemicals and Reagents 29 2 . 1 . 2 VPA Metabolites and Internal Standards 30 2 . 1 . 3 M a t e r i a l s 31 2 . 1 . 4 Animals 31 2.2 Instrumentation 32 2.2.1 Nuclear Magnetic Resonance Spectrometry 32 2 . 2 . 2 Packed Column Gas Chromatography-Mass Spectrometry 32 2 . 2 . 3 C a p i l l a r y Column Gas Chromatography-Mass Spectrometry 33 2 . 2 . 4 Centrifuges 33 2.3 Chemical Syntheses 35 2 . 3 . 1 Synthesis of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d ((E)-2-ene VPA) 35 2 . 3 . 1 . 1 Synthesis of ethyl 2-n-propyl-3-hydroxypentanoate 35 2 . 3 . 1 . 2 Synthesis of 2 - n - p r o p y l - ( E ) - 2 -pentenoic a c i d 36 2 . 3 . 2 Synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d ( ( E , Z ) - 2 , 3 ' - d i e n e VPA) 37 2 . 3 . 2 . 1 Synthesis of ethyl 2 - ( l ' - h y d r o x y p r o p y l ) -(Z)-3-pentenoate 37 vi i 2 . 3 . 2 . 2 Synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d 38 2 . 3 . 3 Synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) -2-pentenoic a c i d ( ( E , E ) - 2 , 3 ' - d i e n e VPA) 40 2 . 3 . 3 . 1 Synthesis of ethyl (Z)-2-pentenoic a c i d 40 2 . 3 . 3 . 2 Synthesis of ethyl 2 - ( l ' - h y d r o x y p r o p y l ) -(E)-3-pentenoate 40 2 . 3 . 3 . 3 Synthesis of ethyl ( E ) - 2 - ( l ' - p r o p e n y l ) -(E)-2-pentenote 41 2 . 3 . 3 . 4 Synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d 42 2.4 Animal Experiments 44 2 . 4 . 1 Pharmacokinetics and Tissue D i s t r i b u t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' -diene VPA 44 2 . 4 . 2 Anticonvulsant Evaluation 45 2 . 4 . 3 P r o t e i n Binding of (E,E) and ( E , Z ) - 2 , 3 ' -diene VPA 46 2.5 A n a l y t i c a l 47 2 . 5 . 1 Homogenization of the Tissue Samples 47 2 . 5 . 2 Internal Standards 48 2 . 5 . 3 C a l i b r a t i o n Curves 48 2 . 5 . 4 E x t r a c t i o n and D e r i v a t i z a t i o n 52 2 . 5 . 5 E x t r a c t i o n E f f i c i e n c y 53 2.6 C a l c u l a t i o n s and S t a t i s t i c s 54 2 . 6 . 1 C a l c u l a t i o n of Pharmacokinetic Parameters 54 2 . 6 . 2 S t a t i s t i c a l A n a l y s i s 54 3 : RESULTS 55 3.1 Chemical Syntheses 55 3.1 .1 2 - n - P r o p y l - ( E ) - 2 - p e n t e n o i c Acid ((E)-2-ene VPA) 55 VI 1 1 3 . 1 . 2 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c Acid ( ( E , Z ) - 2 , 3 ' diene VPA) 55 3 . 1 . 3 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c Acid ( ( E , E ) - 2 , 3 ' - d i e n e VPA) 61 3.2 Assay 66 3 . 2 . 1 E x t r a c t i o n and D e r i v a t i z a t i o n 66 3 . 2 . 2 C a l i b r a t i o n Curves 68 3 . 2 . 3 Detection of VPA and Its Metabolites 68 3.3 Pharmacokinetics and Tissue D i s t r i b u t i o n of VPA 82 3 . 3 . 1 P r o f i l e of VPA i n Plasma 82 3 . 3 . 2 P r o f i l e of VPA i n Peripheral Tissues 82 3 . 3 . 3 P r o f i l e of VPA i n Brain 84 3.4 Pharmacokinetics and Tissue D i s t r i b u t i o n of (E)-2-ene VPA 86 3 . 4 . 1 P r o f i l e of (E)-2-ene VPA i n Plasma 86 3 . 4 . 2 P r o f i l e of (E)-2-ene VPA i n Peripheral Tissues 87 3 . 4 . 3 P r o f i l e of (E)-2-ene VPA i n Brain 87 3 . 5 Pharmacokinetics and Tissue D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA 89 3 . 5 . 1 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Plasma 89 3 . 5 . 2 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Peripheral Tissues 91 3 . 5 . 3 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Brain 93 3.6 Comparative Pharmacokinetics and Tissue D i s t r i b u t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA 95 3 . 6 . 1 P r o f i l e s i n Plasma 95 3 . 6 . 2 P r o f i l e s i n Peripheral Tissues 95 3 . 6 . 3 P r o f i l e s i n Brain 99 3.7 Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Me abolites of VPA 105 ix 3.7 .1 P r o f i l e s of VPA Metabolites i n Plasma 105 3 . 7 . 2 P r o f i l e s of VPA Metabolites i n Peripheral Tissues 109 3 . 7 . 3 P r o f i l e s of VPA Metabolites i n Brain 113 3 . 8 Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Metabolites of (E)-2-ene VPA 116 3 . 8 . 1 P r o f i l e s of (E)-2-ene VPA Metabolites i n Plasma 116 3 . 8 . 2 P r o f i l e s of (E)-2-ene VPA Metabolites i n Peripheral Tissues 116 3 . 8 . 3 P r o f i l e s of (E)-2-ene VPA Metabolites i n Brain 120 3.9 Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA 122 3 . 9 . 1 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Plasma 122 3 . 9 . 2 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Peripheral Tissues 126 3 . 9 . 3 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Brain 130 3.10 Plasma P r o t e i n Binding of ( E , E ) - 2 , 3 ' - d i e n e VPA and ( E , Z ) - 2 , 3 ' - d i e n e VPA 134 3.11 Anticonvulsant Evaluation of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and ( E , Z ) - 2 , 3 ' -diene VPA i n Rats 135 3.11.1 PTZ-Induced Seizure Test 135 3.11.2 Observed Adverse E f f e c t s of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and ( E , Z ) - 2 , 3 ' - d i e n e VPA 140 4 . DISCUSSION 143 4.1 Chemical Syntheses 143 4 . 4 . 1 2 - n - P r o p y l - ( E ) - 2 - p e n t e n o i c Acid 143 4 . 1 . 2 2 - ( ( Z ) - l ' - P r o p e n y l ) - ( E ) - 2 - p e n t e n o i c Acid 145 4 . 1 . 3 2 - ( ( E ) - l ' - P r o p e n y l ) - ( E ) - 2 - p e n t e n o i c Acid 149 4.2 Assay Development f o r VPA and Its Metabolites i n Rat Tissue 151 4.3 Pharmacokinetics and Tissue D i s t r i b u t i o n of VPA i n Rats 158 4 . 3 . 1 P r o f i l e of VPA i n Plasma 158 4 . 3 . 2 P r o f i l e of VPA i n Peripheral Tissues 160 4 . 3 . 3 P r o f i l e of VPA i n Brain 163 4.4 Pharmacokinetics and Tissue D i s t r i b u t i o n of (E)-2-ene VPA i n Rats 167 4 . 4 . 1 P r o f i l e of (E)-2-ene VPA i n Plasma 167 4 . 4 . 2 P r o f i l e of (E)-2-ene VPA i n Peripheral Tissues 170 4 . 4 . 3 P r o f i l e of (E)-2-ene VPA i n Brain 171 4 . 4 . 4 Evaluating the Differences Between (E)-2-ene VPA and VPA i n Rats 172 4 . 5 Pharmacokinetics and Tissue D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Rats 173 4 . 5 . 1 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Plasma 173 4 . 5 . 2 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Peripheral Tissues 175 4 . 5 . 3 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Brain 176 4 . 5 . 4 Evaluating Differences i n the Tissue D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA to VPA and (E)-2-ene VPA 178 4 . 6 Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Metabolites of VPA i n Rats 179 4 . 6 . 1 P r o f i l e s of VPA Metabolites i n Plasma 179 4 . 6 . 2 P r o f i l e of VPA Metabolites i n Peripheral Tissues 181 4 . 6 . 3 P r o f i l e of VPA Metabolites i n Brain 182 4.7 Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Metabolites of (E)-2-ene VPA i n Rats 185 4.7 .1 P r o f i l e s of (E)-2-ene VPA Metabolites i n Plasma 4 . 7 . 2 P r o f i l e s of (E)-2-ene VPA Metabolites i n Peripheral Tissues 4 . 7 . 3 P r o f i l e s of (E)-2-ene VPA Metabolites i n Brain 8 . Tissue D i s t r i b u t i o n and K i n e t i c P r o f i l e s of Metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Rats 4 . 8 . 1 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Plasma 4 . 8 . 2 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Peripheral Tissues 4 . 8 . 3 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Brain 9 Anticonvulsant Evaluation of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and ( E , Z ) - 2 , 3 ' -diene VPA i n Rats SUMMARY AND CONCLUSIONS REFERENCES APPENDICES LIST OF FIGURES gure Chemical s t r u c t u r e s of some commonly used anticonvulsant agents. Chemical s t r u c t u r e s of two known hepatotoxic agents 4-pentenoic a c i d and methylenecyclopropylacetic a c i d and two metabolites of VPA suspected of being r e s p o n s i b l e f o r VPA h e p a t o t o x i c i t y . Metabolic pathways of v a l p r o i c a c i d : (a) w - o x i d a t i o n , (b) dehydrogenation, (c) g l u c u r o n i d a t i o n , and (d) (w-1)-o x i d a t i o n . The 0 - o x i d a t i o n pathway of v a l p r o i c a c i d . Total ion chromatogram and mass spectra of methyl 2-n-propyl-(E)-2-pentenoate and methyl 2 - n - p r o p y l - ( Z ) - 2 -pentenoate. 400 MHz Proton NMR spectrum of 2 - n - p r o p y l - ( E ) - 2 -pentenoic a c i d (*=2-n-propyl-(Z)-2-pentenoic a c i d ) . Total ion chromatogram and mass spectrum of methyl 2 - ( ( Z ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o a t e . 400 MHz Proton NMR spectrum of 2 - ( ( Z ) - l ' - p r o p e n y l ) -(E)-2-pentenoic a c i d ( * = 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d ) . Total ion chromatogram and mass spectrum of methyl 2 - ( ( E ) - 1 ' - p r o p e n y l ) - (E)-2-pentenoate. 300 MHz Proton NMR spectrum of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) -2-pentenoic a c i d ( * = 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d ) . High and low range concentration c a l i b r a t i o n curves f o r VPA i n brain t i s s u e homogenate. High and low range concentration c a l i b r a t i o n curves f o r ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t b r a i n t i s s u e homogenate. High and low range concentration c a l i b r a t i o n curves f o r ( E , Z ) - 2 , 3 ' - d i e n e VPA i n r a t brain t i s s u e homogenate. C a l i b r a t i o n curve f o r ( E ) - 2 , 4 - d i e n e VPA i n r a t brain t i s s u e homogenate. xi i i 15 High and low range concentration c a l i b r a t i o n curves f o r (E)-2-ene VPA i n b r a i n t i s s u e homogenate. 73 16 High and low range concentration c a l i b r a t i o n curves f o r (Z)-2-ene VPA i n r a t brain t i s s u e homogenate. 74 17 C a l i b r a t i o n curves f o r 3-ene VPA and 4-ene VPA i n r a t b r a i n t i s s u e homogenate. 75 18 C a l i b r a t i o n curves f o r 3-keto VPA and 4-keto VPA i n r a t b r a i n t i s s u e homogenate. 76 19 C a l i b r a t i o n curves f o r 3-OH VPA and 4-OH VPA i n r a t b r a i n t i s s u e homogenate. 77 20 C a l i b r a t i o n curves f o r 5-OH VPA and 2-PGA i n r a t brain t i s s u e homogenate. 78 21 Mass chromatograms of VPA, VPA m e t a b o l i t e s , and i n t e r n a l standards i n r a t brain t i s s u e homogenate. 81 22 VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . 83 23 VPA concentration-time curves i n r a t plasma, whole brain (WB), hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), and putamen (CP) f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (WB,n=8/time p o i n t ; brain regions were pooled,n=8/time p o i n t ) . 85 24 (E)-2-ene VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . 88 25 (E)-2-ene VPA concentration-time curves i n r a t plasma, whole b r a i n (WB), hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), substantia n i g r a (SN), medulla (MED), and putamen (CP) f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (WB,n=8/time p o i n t ; brain regions were pooled,n=8/time p o i n t ) . 90 26 ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . 92 xiv 27 ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n r a t plasma, whole b r a i n (WB), hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), substantia n i g r a (SN), medulla (MED), and putamen (CP) f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (WB,n=8/time p o i n t ; brain regions were pooled,n=8/time p o i n t ) . 94 28 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n plasma f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 96 29 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n l i v e r f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 100 30 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n kidneys f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 101 31 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n - t i m e curves i n heart f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 102 32 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n - t i m e curves i n lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 103 33 VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n whole brain f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 104 34 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 108 35 Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 110 36 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . I l l XV 37 Concentration-time p l o t s of VPA and i t s polar metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 112 38 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled/time p o i n t ) . 114 39 Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled/time p o i n t ) . 115 40 Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 117 41 Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 118 42 Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 119 43 Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 121 44 Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8 pooled/time p o i n t ) . 123 45 Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8 pooled/time p o i n t ) . 124 46 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 5% ( E , E ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 125 XVI 47 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/titne p o i n t , S.D. omitted f o r c l a r i t y ) . 127 48 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA ( c o n t a i n i n g 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 128 49 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 129 50 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n whole brain f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 131 51 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E ,E)-2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8 pooled/time p o i n t ) . 132 52 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA ( c o n t a i n i n g 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8 pooled/time p o i n t ) . 133 53 Binding isotherm of ( E , E ) - 2 , 3 ' - d i e n e VPA and (E,Z)-2 , 3 ' - diene VPA i n r a t plasma as determined by u l t r a c e n t r i f u g a t i o n (n=8). 137 54 Dose-response curves f o r VPA, (E)-2-ene VPA, (E ,Z)-2 , 3 ' - d i e n e VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA f o l l o w i n g 150 mg/kg i - P - a d m i n i s t r a t i o n of each compound to r a t s (n=8/point). 139 55 Chemical s t r u c t u r e s of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d and 2 - n - p r o p y l - ( Z ) - 2 - p e n t e n o i c a c i d i l l u s t r a t i n g the s h i e l d i n g and d e s h i e l d i n g e f f e c t s of the v i n y l i c protons. 146 56 Chemical s t r u c t u r e s of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d and 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d i l l u s t r a t i n g the s h i e l d i n g and d e s h i e l d i n g e f f e c t s of the v i n y l i c protons. 148 xvi i 57 Mass chromatogram at m/z 139 of t-BDMS d e r i v a t i v e s of (A) peak l = ( E , Z ) - 2 , 3 ' - d i e n e VPA and peak 2=(E,E)-2 , 3 ' - d i e n e VPA i n control human serum; (B) peak 3= ( Z ) - 2 , 4 - d i e n e VPA and peak 4=(E)-2,4-diene VPA i n c o n t r o l human serum; (C) serum sample of a p a t i e n t on VPA therapy containing peaks 1 to 4, on an 0V-1701 column. 152 58 Mass chromatogram at m/z 139 of PFB d e r i v a t i v e s of (A) peak l = ( E , Z ) - 2 , 3 ' - d i e n e VPA and peak 2=(E,E)-2 , 3 ' - d i e n e VPA i n c o n t r o l human serum; (B) peak 3= ( Z ) - 2 , 4 - d i e n e VPA and peak 4=(E)-2,4-diene VPA i n c o n t r o l human serum; (C) serum sample of a p a t i e n t on VPA therapy containing peaks 1 to 4 , on a DB-1 column. 154 59 General e x t r a c t i o n and d e r i v a t i z a t i o n scheme f o r the NICI GC-MS a n a l y s i s of VPA and VPA metabolites i n r a t plasma and homogenized t i s s u e . 156 60 Mass chromatogram at m/z 139 of ( E , Z ) - 2 , 3 ' - d i e n e VPA ( a ) , ( E ) - 2 , 4 - d i e n e VPA ( b ) , and ( E , E ) - 2 , 3 ' - d i e n e VPA (c) i n l i v e r homogenate. 157 61 Tissue/plasma r a t i o s of VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA i n whole brain (WB), l i v e r (LIV) , kidneys (KID), heart (HEA), and lungs (LUN) of r a t s at t m a x and tjQh f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound (n=8,error bars=S.D.) . 162 62 Concentrations of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' -diene VPA i n hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), putamen (CP), whole brain (WB), and (PLA) of r a t s at t m a x and tjoh f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound (WB&PLAn=8,HIP...CP pooled from 8 r a t s ) . 165 63 Tissue/plasma r a t i o s of VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA i n hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), putamen (CP), whole b r a i n (WB), and plasma (PLA) of r a t s at t m a x and tjoh f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound (WB&PLA n=8,HIP. . .CP pooled from 8 r a t s ) . 166 64 65 Proposed metabolic scheme of (E)-2-ene VPA i n r a t s Proposed metabolic scheme of ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s 187 191 LIST OF TABLES A summary of the two main types of e p i l e p s i e s and t h e i r synonyms. The drugs of choice f o r both generalized and l o c a l i z a t i o n - r e l a t e d s e i z u r e s . Mass-to-charge r a t i o s (m/z) f o r the i n t e r n a l standards ( * ) , VPA and VPA m e t a b o l i t e s . Volumes of c o n t r o l t i s s u e homogenate used f o r the d i l u t i o n of stock t i s s u e homogenate containing VPA and VPA metabolites to provide the samples required to produce a c a l i b r a t i o n curve. Concentrations f o r the low and high c a l i b r a t i o n curves f o r VPA and i t s m e t a b o l i t e s . The e x t r a c t i o n e f f i c i e n c i e s of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA from brain t i s s u e homogenate at various concentrations (n=3). Inter-assay v a r i a t i o n based on the slopes of the c a l i b r a t i o n s curves over an 18 month period f o r VPA and i t s m e t a b o l i t e s . ()=n Ions monitored f o r the q u a n t i t a t i v e a n a l y s i s and the r e t e n t i o n times of i n t e r n a l s t a n d a r d s ( * ) , VPA and i t s m e t a b o l i t e s . The time to peak ( t m a x ) and peak concentration ( C m a x ) i n t i s s u e s and plasma f o l l o w i n g VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n . AUC 0 to 10 h i n t i s s u e s f o l l o w i n g i . p . a d m i n i s t r a t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA. The time to peak ( t m a x ) and peak concentration ( C m a x ) i n b r a i n t i s s u e s f o l l o w i n g e i t h e r VPA, (E)-2-ene VPA, or ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n . AUC from 0 to 10 hours i n the i n d i v i d u a l pooled b r a i n s e c t i o n s f o l l o w i n g i . p . a d m i n i s t r a t i o n of e i t h e r VPA, (E)-2-ene VPA, or ( E , E ) - 2 , 3 ' - d i e n e VPA. Plasma p r o t e i n binding of ( E , E ) - 2 , 3 ' - d i e n e VPA and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n rat plasma by u l t r a c e n t r i f u g a t i n=8 ( S . D . ) . The percent of r a t s protected from PTZ-induced s e i z u r e s a f t e r the i . p . a d m i n i s t r a t i o n of 70 mg/kg s . c . of PTZ 30 minutes post drug (n=8/dose). The mean e f f e c t i v e doses against PTZ-induced s e i z u r e s and the slopes of the log dose-response p l o t s f o r each compound tested i n r a t s . XX LIST OF SCHEMES Scheme Page 1 Synthesis of (E)-2-ene VPA: a=EtOH, H2SO4, benzene, A; b=[(CH3)2CH]2NH, n - B u L i , CH3CH2CHO, THF, - 7 8 ° C ; c=(C 2 H 5) 3N, CH3S02C1, CH 2 C1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, HCl . 56 2 Synthesis of ( E , Z ) - 2 , 3 ' - d i e n e VPA: a=EtOH, H2S04, benzene, A; b=[(CH3)2CH]2NH, n - B u L i , CH3CH2CHO, THF, - 7 8 ° C ; c=(C 2 H 5) 3N, CH3S02C1, CH 2 C1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, HCl. 59 3 Synthesis of ( E , E ) - 2 , 3 ' - d i e n e VPA: a=K2C03 , E t I , 18-crown-6, THF, A; b=[(CH3)2CH]2NH, n - B u L i , HMPA, CH3CH2CH0, THF, - 7 8 ° C ; c=(C 2 H5) 3N, CH3S02C1, CH 2 C1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, H C l . 63 X X I SYMBOLS AND ABBREVIATIONS AUC area under the curve bp b o i l i n g point CC corpus callosum CER cerebellum CI chemical i o n i z a t i o n Cl clearance C m a x peak drug concentration CNS c e n t r a l nervous system CP putamen CSF cerebral s p i n a l f l u i d d doublet DBU l , 8 - d i a z a b i c y c l o [ 5 . 4 . 0 . ] u n d e c - 7 - e n e dd doublet of doublets dt doublet of t r i p l e t s E trans EI e l e c t r o n impact FFA f r e e f a t t y a c i d GABA gamma aminobutyric a c i d GABA-t gamma aminobutyric acid-transaminase GAD g l u t a r i c a c i d decarboxylase GC-MS gas chromatography-mass spectrometry *H proton HIP hippocampus i . d . i n t e r n a l diameter xxi i IC i n f e r i o r c o l l i c u l u s i . p . i n t r a p e r i t o n e a l i . v . intravenous J coupling constant LES low frequency electroshock s t i m u l a t i o n l i t . l i t e r a t u r e m meter/multiplet M+ molecular ion MED medulla MES maximum electroshock seizures MHz megahertz mmoles m i l l i m o l e s MSTFA N - m e t h y l - N - t r i m e t h y l s i l y l t r i f l u o r o acetamide MW molecular weight m/z mass/charge NICI negative ion chemical i o n i z a t i o n NMR nuclear magnetic resonance OLF o l f a c t o r y bulbs PFB pentafluorobenzyl ppm parts per m i l l i o n PTZ pentylenetetrazole q quartet s . c . subcutaneous SC s u p e r i o r c o l l i c u l u s SN s u b s t a n t i a n i g r a t t r i p l e t t-BDMS t e r t i a r y - b u t y l d i m e t h y l s i 1ane THF tetrahydrofuran TIC t o t a l ion chromatogram tmax time to reach peak drug concentration TMS t r i m e t h y l s i lane VPA v a l p r o i c a c i d [2H]6VPA [ 2 H ] s v a l p r o i c a c i d WB whole brain Z c i s 2 , 3 ' - d i e n e VPA 2 - ( l ' - p r o p e n y l ) - 2 - p e n t e n o i c a c i d 2 , 4 - d i e n e VPA 2 - n - p r o p y l - 2 , 4 - p e n t a d i e n o i c a c i d 2-ene VPA 2-n-propyl-2-pentenoic a c i d [ 2 H] 3 2-ene VPA [ t H ] 3 2 - n - p r o p y l - 2 , 4 - p e n t a d i e n o i c a c i d 3-ene VPA 2 - n - p r o p y l - 3 - p e n t e n o i c a c i d 4-ene VPA 2-n-propyl-4-pentenoic a c i d 3-keto VPA 2-n-propyl-3-oxopentanoic a c i d [ 2 H ] 3 3 - k e t o VPA [ 2 H]32-n-propyl-3-oxopentanoic a c i d 4-keto VPA 2-n-propyl-4-oxopentanoic a c i d 3-OH VPA 2-n-propyl-3-hydroxypentanoic a c i d 4-OH VPA 2-n-propyl-4-hydroxypentanoic a c i d 5-OH VPA 2-n-propyl-5-hydroxypentanoic a c i d 2-MGA 2 - m e t h y l g l u t a r i c a c i d 2-PGA 2 - p r o p y l g l u t a r i c a c i d 2-PMA 2-propylmalonic a c i d 2-PSA 2 - p r o p y l s u c c i n i c a c i d ACKNOWLEDGEMENTS I am s i n c e r e l y g r a t e f u l to my s u p e r v i s o r , Dr. Frank Abbott, f o r h i s continued support, encouragement, e x c e l l e n t guidance, and f r i e n d s h i p throughout the course of my s t u d i e s . The author would l i k e to thank the members of the supervisory committee, Drs. James E. Axelson, W i l l i a m Godolphin, Marc Levine, Keith McErlane, and James M. Orr , f o r t h e i r time and h e l p f u l c o n t r i b u t i o n s towards the f i n a l i z i n g of t h i s t h e s i s . I would a l s o l i k e to extend my thanks to Mr. Roland Burton f o r h i s t e c h n i c a l e x p e r t i s e ; to my colleagues Dr. Kelem Kassahun, Anthony B o r e l , Dr. Abdul M u t l i b , Matthew Wright, and J i a o j i a o Zheng f o r t h e i r h e l p f u l advice and support; and to Mr. V i c t o r Wang f o r h i s help with the animal experiments. F i n a n c i a l support was provided by the Faculty of Pharmaceutical Sciences and the U n i v e r s i t y of B r i t i s h Columbia and i s g r a t e f u l l y acknowledged. XXV To my l o v i n g wife and c h i l d r e n f o r t h e i r unflagging support, encouragement, and patience throughout my s t u d i e s . 1. INTRODUCTION 1.1 VALPROIC ACID 1.1.1 Overview Epilepsy i s a common d i s o r d e r occurring i n at l e a s t 1 out of 200 people i n the U.S. (Lechtenberg, 1990). Epilepsy cannot be defined as a s i n g l e disease but rather as a group of d i s o r d e r s having s i m i l a r p a t h o p h y s i o l o g i c a l p r o p e r t i e s (Lechtenberg, 1990). In f a c t , the term e p i l e p t i c syndrome would be more appropriate rather than d i s e a s e , since only a few diseases have been associated with e p i l e p s y (Dreifuss et al., 1985). An e p i l e p t i c syndrome i s a group of signs and symptoms ( c l i n i c a l or a n c i l l a r y ) o c c u r r i n g together and may not have a common e t i o l o g y or prognosis (Dreifuss et a 7 . , 1985; Levy et a / . , 1989). More s p e c i f i c a l l y e p i l e p s y i s a symptom of excessive neuronal discharges r e s u l t i n g from e i t h e r e x t r a or i n t r a c r a n i a l d i s t u r b a n c e s . The two common types of e p i l e p s i e s are p a r t i a l and generalized s e i z u r e s , although other terms have been used i n the l i t e r a t u r e (table l ) ( D r e i f u s s et a / . , 1985; Levy et a 7 . , 1989). The i n i t i a l protocol f o r the treatment of an e p i l e p t i c episode i s nearly always chemically based. During the nineteenth century, bromide s a l t s were introduced f o r the management of seizures but were l a t e r replaced i n 1912 by phenobarbital (Lechtenberg, 1990). In 1938 phenytoin was marketed and q u i c k l y became the benchmark f o r future a n t i e p i l e p t i c s . The majority of the drugs used to manage e p i l e p s y today are s t r u c t u r a l l y s i m i l a r to 2 Table 1: A summary of the two main types of e p i l e p s i e s and t h e i r synonyms (Dreifuss et al., 1985). PARTIAL SEIZURES GENERALIZED SEIZURES f o c a l s e i z u r e s primary e p i l e p s y l o c a l i z a t i o n a l - r e l a t e d e p i l e p s y i d i o p a t h i c e p i l e p s y secondary e p i l e p s y cryptogenic e p i l e p s y symptomatic e p i l e p s y phenobarbital or phenytoin a l b e i t with one e x c e p t i o n , v a l p r o i c a c i d (VPA)(figure 1). VPA i s an eight carbon branched chain f a t t y a c i d synthesized i n i t i a l l y i n 1881 f o r use as an organic s o l v e n t . In 1963 VPA was a c c i d e n t l y discovered to have anticonvulsant a c t i v i t y i n animals (Meunier et al., 1963). Soon a f t e r (1967) VPA was introduced i n t o Europe as an a n t i e p i l e p t i c agent and became a v a i l a b l e i n the United States by 1978. In 1983 VPA was marketed as a syrup and g e l a t i n capsule under the trade name Depakene^ and since then has been used e x t e n s i v e l y f o r the successful management of primary generalized and some p a r t i a l seizures ( t a b l e 2)(Dulac and A r t h u i s , 1984; Chadwick, 1987; Bourgeois et al., 1987; W i l d e r , 1987; Dean and Penry, 1988). Associated with VPA treatment i s a delay i n the onset of maximal anticonvulsant a c t i v i t y (Jeavons and C l a r k , 1974; Rowan et al., 1979) together with an extended duration of a c t i v i t y even a f t e r the disappearance of the parent compound from the c i r c u l a t i o n i n both humans and animals (Lockard and Levy, 1976; Harding et al., 1978). I t appears that the concentration of VPA i n plasma does not correspond with anticonvulsant a c t i v i t y . Since VPA i s e x t e n s i v e l y metabolized by the l i v e r , perhaps one or more of these metabolites would be a b e t t e r measure of anticonvulsant a c t i v i t y . The present study w i l l i n v e s t i g a t e the p o t e n t i a l c o n t r i b u t i o n of a major VPA m e t a b o l i t e , ( E , E ) - 2 , 3 ' - d i e n e VPA, to these unusual pharmacodynamics of VPA. 1 .1 .2 Adverse Reactions Adverse r e a c t i o n s of VPA can be c l a s s i f i e d i n t o types A, B, and t e r a t o g e n i c e f f e c t s (Dreifuss and Langer, 1988). The type A r e a c t i o n s COOH Valproic Acid Figure 1: Chemical s t r u c t u r e s of some commonly used anticonvulsant agents. 5 Table 2: The drugs of choice f o r both generalized and l o c a l i z a t i o n -r e l a t e d s e i z u r e s . F i r s t Second Choice Choice Generalized Seizures I d i o p a t h i c (Primary) absence VPA/ESM CZP a t y p i c a l VPA CZP myoclonic VPA PB t o n i c VPA DPH c l o n i c VPA DPH t o n i c - c l o n i c VPA DPH a t o n i c VPA DPH L o c a l i z a t i o n a l - R e l a t e d ( P a r t i a l ) Seizures i d i o p a t h i c CBZ/DPH PRM/VPA/PB (simple and complex) secondary g e n e r a l i z e d CBZ/DPH VPA/PRM/PB VPA=valproic a c i d ; CZP=clonazepam; ESM=ethosuximide; PB=phenobarbital; DPH=phenytoin; CBZ=carbamazepine; PRM=primidone; are d o s e - r e l a t e d and u s u a l l y tend to occur at the upper end of the dose-response curve. Type A r e a c t i o n s w i l l occur i n most p a t i e n t s i f the dose i s high enough; however, the side e f f e c t s u s u a l l y subside when the dose i s lowered. In g e n e r a l , a type A adverse r e a c t i o n i s not l i f e threatening unless a f a t a l underlying disease i s involved or the drug i s taken i n an overdose s i t u a t i o n . In c o n t r a s t , a type B adverse r e a c t i o n i s u n p r e d i c t a b l e , i s not considered d o s e - r e l a t e d , and may be f a t a l . The causes of a type B r e a c t i o n are u s u a l l y unknown ( i d i o s y n c r a t i c ) ; however, the r e a c t i o n sometimes involves a metabolic d i s o r d e r (Zimmerman and Ishak, 1982; Dreifuss and Langer, 1988). Teratogenic e f f e c t s can occur with a therapeutic and non-toxic dose to the mother but exposure of the fetus to VPA can r e s u l t i n a host of developmental c o m p l i c a t i o n s . 1 .1 .2 .1 Type A Adverse E f f e c t s of VPA G a s t r o i n t e s t i n a l side e f f e c t s are the most common complaints from p a t i e n t s on VPA. Up to 50% of p a t i e n t s on VPA therapy experience g a s t r o i n t e s t i n a l disturbances (nausea, v o m i t i n g , anorexia) that can be managed by reducing the dose and/or by taking the drug with food (Wilder et al., 1983; Bruni and W i l d e r , 1979). Central nervous system side e f f e c t s such as sedation can occur i n 50% of the p a t i e n t s on i n i t i a l VPA treatment but a f t e r long-term use l e s s than 1% of p a t i e n t s are a f f e c t e d by drowsiness (Jeavons et al., 1977; Gram et al., 1977; Lance and Anthony, 1977). Fine tremor has been a s s o c i a t e d with VPA therapy but t h i s symptom can be a l l e v i a t e d by a d j u s t i n g the dose or dosing regime such that the amplitude of the peak and trough plasma concentrations are reduced (Hyman et al, 1979). Other adverse e f f e c t s of VPA that are not as common and r e l a t i v e l y l e s s traumatic are weight gain (Dinessen et al, 1984), s k i n rash (Bruni and A l b r i g h t , 1983), and h a i r changes (depigmentation, a l o p e c i a , perming effect)(Rauskanen et al., 1979; Gupta, 1988). 1.1.2.2 Type B Adverse E f f e c t s of VPA: H e p a t o t o x i c i t y The i d i o s y n c r a t i c h e p a t o t o x i c i t y associated with VPA therapy was i n i t i a l l y thought to be a rare but f a t a l r e a c t i o n with an o v e r a l l incidence of 1:10000. As the use of VPA increased so d i d the rate of deaths, with an incidence of h e p a t o t o x i c i t y reported to be as low as 1:500 f o r high r i s k p a t i e n t s (Dreifuss et al, 1989). Today the number of f a t a l i t i e s has decreased d r a m a t i c a l l y since c l i n i c i a n s are becoming i n c r e a s i n g l y aware of the r i s k f a c t o r s f o r h e p a t o t o x i c i t y associated with VPA. Fulminating h e p a t o t o x i c i t y associated with VPA use u s u a l l y occurs w i t h i n the f i r s t 4 to 6 months of therapy, with c h i l d r e n under the age of 10 years representing 73% of a l l f a t a l i t i e s (Dreifuss et al., 1987). At highest r i s k were p a t i e n t s l e s s than 2 years of age on m u l t i -anticonvulsant treatment, i.e. VPA-polytherapy. For those p a t i e n t s over 2 years of age on VPA-monotherapy, the r i s k of developing f a t a l h e p a t o t o x i c i t y has decreased to 1:37000 (Dreifuss et al., 1989). Some of the common p a t h o l o g i c a l f i n d i n g s from p a t i e n t s e x h i b i t i n g VPA-induced h e p a t o t o x i c i t y are increased body temperature, elevated l i v e r enzymes, j a u n d i c e , hepatic m i c r o v e s i c u l a r s t e a t o s i s and necrosis with mitochondrial d i s s o l u t i o n , increased i n t r a c r a n i a l p r e s s u r e , and coma (Gerber et al., 1979; Itoh et al., 1982; Zimmerman and Ishak, 1982; Scheffner et al., 1988; Sugimoto et al., 1987). Although the mechanism of VPA h e p a t o t o x i c i t y i s not known, there are s i m i l a r i t i e s to Reye's Syndrome, hypoglycin t o x i c i t y (Jamaican Vomiting Sickness) and 4-pentenoic a c i d t o x i c i t y (Kesterson et al., 1984). Both 4-pentenoic a c i d and methylenecyclopropylacetic a c i d (a metabolite of hypoglycin) are known steatogenic agents (Zimmerman and Ishak, 1982). Two metabolites of VPA, namely 4-ene VPA and 2,4-diene VPA are s t r u c t u r a l l y s i m i l a r to 4-pentenoic a c i d and methylenecyclopropylacetic a c i d ( f i g u r e 2 ) . Upon GC-MS a n a l y s i s of the urine and plasma samples from p a t i e n t s succumbing to VPA-induced h e p a t o t o x i c i t y , abnormally high l e v e l s of 4-ene VPA and (E)-2 , 4 - d i e n e VPA and decreased l e v e l s of 3-keto VPA and 3-OH VPA, products of /3-oxidation, were discovered (Scheffner et al., 1988). Evidence i n humans, such as mitochondrial damage and a decrease i n the ^ - o x i d a t i o n products, 3-keto VPA and 3-OH VPA, i n d i c a t e d that /3-oxidation was being i n h i b i t e d . An i n h i b i t i o n of ^ - o x i d a t i o n w i l l r e s u l t i n a shunting of VPA metabolism to other pathways. This could e x p l a i n the higher l e v e l s of 4-ene VPA and 2 , 4 - d i e n e VPA found i n the plasma and urine of hepatotoxic p a t i e n t s , since the o r i g i n of these metabolites i s via microsomal metabolism. Therefore, the d i s r u p t i o n or i n t e r f e r e n c e of f a t t y a c i d /J-o x i d a t i o n by an unknown f a c t o r could be a prelude to VPA h e p a t o t o x i c i t y . I s o l a t e d r a t hepatocytes showed a d o s e - r e l a t e d t o x i c i t y to VPA (Kingsley et al., 1980). Rats administered a s i n g l e dose of VPA showed a l t e r e d hepatocytes and mitochondria (Jezequel et al., 1984). Rats administered e i t h e r 4-ene VPA or 2 , 4 - d i e n e VPA were found to develop f a t t y l i v e r s , m i c r o v e s i c u l a r s t e a t o s i s and n e c r o s i s , and mitochondrial damage s i m i l a r to that observed i n p a t i e n t s d i s p l a y i n g VPA h e p a t o t o x i c i t y (Kesterson et al., 1984). The cause of VPA-induced h e p a t o t o x i c i t y i s thought to involve p o s s i b l e r e a c t i v e intermediates generated by common biotransformation C O O H 4 - E N E V A L P R O I C ACID C O O H 2 . 4 - D I E N E V A L P R O I C ACID C O O H 4 - P E N T E N O I C ACID C O O H M E T H Y L E N E C Y C L O -P R O P Y L A C E T I C ACID Chemical structures of two known hepatotoxic agents 4-pentenoic acid and methylenecyclopropylacetic acid and two metabolites of VPA suspected of being responsible for VPA hepatotoxicity. processes. The metabolism of the hepatotoxic agent, 4-ene VPA, by cytochrome P-450 or ^ - o x i d a t i o n to y i e l d a p o t e n t i a l e l e c t r o p h i l i c species i s c u r r e n t l y being i n v e s t i g a t e d ( B a i l l i e , 1988). The metabolism of 4-ene VPA by the l i v e r microsomal cytochrome P-450 enzymes generated a f r e e r a d i c a l intermediate that could a l k y l a t e heme, thereby destroying the mixed-function oxidase ( O r t i z de Montellano and C o r r e i a , 1983). This route of 4-ene VPA metabolism i s akin to a s u i c i d e - s u b s t r a t e . In a d d i t i o n , the f r e e r a d i c a l intermediate can give r i s e to an epoxide that could p o s s i b l y a l k y l a t e key metabolizing enzymes ( B a i l l i e , 1988). The b i o a c t i v a t i o n of 4-ene VPA v i a mitochondrial ^ - o x i d a t i o n to a r e a c t i v e metabolite was based on knowledge of 4-pentenoic a c i d metabolism (Schulz , 1983). B e t a - o x i d a t i o n of 4-pentenoic a c i d r e s u l t s i n the formation of a h i g h l y r e a c t i v e e l e c t r o p h i l e , 3-oxo-4-pentenoyl-CoA. This r e a c t i v e species can a l k y l a t e 3-ketoacyl-CoA t h i o l a s e , the terminal enzyme i n the ^ - o x i d a t i o n pathway, and thereby i n h i b i t /3-oxidation. The 4-ene VPA could undergo a s i m i l a r f a t e leading to a corresponding e l e c t r o p h i l i c s p e c i e s , 3-oxo-4-ene VPA (Porubek et a / . , 1989). However, from i s o l a t e d perfused r a t l i v e r challenged with 4-ene VPA, 3-0H-4-ene VPA and 2 , 4 - d i e n e VPA were the only metabolites i d e n t i f i e d (Rettenmeier et al., 1985). The authors suggested that f a i l u r e to detect the 3-oxo-4-ene VPA was i n part due to the h i g h l y r e a c t i v e nature of t h i s compound. Current work by Kassahun and Abbott (1989) suggests that the 3-oxo-4-ene VPA may not be the agent r e s p o n s i b l e f o r VPA h e p a t o t o x i c i t y . The metabolite found i n the hepatotoxic p a t i e n t s , ( E ) - 2 , 4 - d i e n e VPA may be the suspect compound. Although s t i l l s p e c u l a t i v e , the conjugation of 2 , 4 - d i e n e VPA with CoA to y i e l d 2 , 4 - d i e n e v a l p r o y l CoA could proceed to a p o t e n t i a l l y r e a c t i v e intermediate upon resonance rearrangement. This r e a c t i v e e l e c t r o p h i l e has the p o t e n t i a l of i n t e r f e r i n g with l i v e r metabolism and u l t i m a t e l y leading to severe h e p a t o t o x i c i t y . 1 . 1 . 2 . 3 Teratogenic E f f e c t s of VPA The t e r a t o g e n i c e f f e c t s of VPA encompass a host of malformations that include f a c i a l anomalies of the nose, l i p s , p a l a t e , eyes, and e a r s ; d i g i t a l anomalies such as p o l y d a c t y l i s m and hypoplasia of the phalanges and n a i l s ; congenital heart defects such as ductus a r t e r i o s u s and v e n t r i c u l a r septal d e f e c t s ; and neural tube defects seen as lumbosacral meningocele and spina b i f i d a (Nau et al., 1982; Kaneko et al., 1988; Carter and Stewart, 1989; B e r t o l l i n i et al., 1985). Approximately 1% of fetuses exposed to VPA in utero develop spina b i f i d a (Nau and Loscher, 1984). U n f o r t u n a t e l y , the a l t e r n a t i v e s are e i t h e r to use other a n t i c o n v u l s a n t s , which are a l s o t e r a t o g e n i c , or to d i s c o n t i n u e drug use and r i s k s e i z u r e s . Animal studies have shown that 4-ene VPA displayed e n a n t i o s e l e c t i v e t e r a t o g e n i c i t y with the S-enantiomer being more t e r a t o g e n i c than VPA (Hauck and Nau, 1989). Conversely, the metabolite (E)-2-ene VPA was found to be l e s s t e r a t o g e n i c than the parent drug when administered at comparable doses suggesting that perhaps (E)-2-ene VPA could be used i n place of VPA when the r i s k of t e r a t o g e n i c i t y becomes relevant (Nau and Loscher, 1984). 1.1 . 3 Metabolism The metabolism of VPA i s h i g h l y complex f o r such a simple molecule ( f i g u r e s 3 and 4 ) . In mammals, the f a t e of VPA i s mainly hepatic metabolism, since only 1 - 3% of the dose i s excreted unchanged COOH (E) -2 ,4-d iene VPA Figure 3: Metabolic pathways of v a l p r o i c a c i d : (a) o - o x i d a t i o n , (b) dehydrogenation, (c) g l u c u r o n i d a t i o n , and (d) ( w - l ) - o x i d a t i o n . COOH (E,E)-2,3'-diene VPA 3-keto VPA + COOH (E,Z)-2,3'-diene VPA Figure 4: The ^ - o x i d a t i o n pathway of v a l p r o i c a c i d . i n the urine (Bruni and W i l d e r , 1979; Gugler and von Unruh, 1980; B a i l e r et al., 1985). Although several metabolic pathways e x i s t , the two major routes are g l u c u r o n i d a t i o n and ^ - o x i d a t i o n with the r e l a t i v e c o n t r i b u t i o n of the l a t t e r occurring to a greater extent i n man when compared to r a t s (Granneman et al., 1984). Minor routes of VPA metabolism i n both man and animals include w - o x i d a t i o n , ( w - l ) - o x i d a t i o n , dehydrogenation, r e d u c t i o n , g l y c i n e conjugation, e p o x i d a t i o n , and i s o m e r i z a t i o n (Granneman et al., 1984; Abbott et al., 1986). In humans, g l u c u r o n i d a t i o n of VPA accounts f o r 10.8 - 68.3% of the dose (Bonora et al., 1979; Bruni and W i l d e r , 1979; B a i l e r et al., 1985; Dickinson et al., 1989). As the dose of VPA i n c r e a s e s , the drug i s shunted away from the /3-oxidation pathway and more towards glucuronide conjugation thereby producing a net increase i n g l u c u r o n i d a t i o n (Granneman et al., 1984). The second major route of VPA metabolism i s ^ - o x i d a t i o n ( f i g u r e 4 ) . Metabolites generated v i a ^ - o x i d a t i o n are (E)-2-ene VPA, 3-OH VPA, and 3-keto VPA (Granneman et al., 1984) and, i n t o t a l , account f o r approximately 12.5% of VPA i n serum (Abbott et al,, 1986; Dickinson et al., 1989; Kassahun et al., 1990). However, the use of (E)-2-ene VPA, 3-0H VPA, and 3-keto VPA to estimate the degree of VPA /3-oxidation could be m i s l e a d i n g . Several of the subsequent products from the ^ - o x i d a t i v e metabolites of VPA occur endogenously as products of FFA metabolism. This c o u l d , i n p a r t , e x p l a i n why 30% of the dose of VPA administered could not be accounted f o r on a mass balance basis using VPA metabolites (Granneman et al., 1984). Products of w-oxidation ( f i g u r e 3) are 5-0H VPA, 2 - p r o p y l g l u t a r i c a c i d (2-PGA), and 2-propylmalonic a c i d (2-PMA) while ( w - l ) - o x i d a t i o n r e s u l t s i n 4-OH VPA, 4-keto VPA, and 2 - p r o p y l s u c c i n i c a c i d (2-PSA)(Granneman et al., 1984). Dehydrogenation i s thought to be responsible f o r the occurrence of several unsaturated metabolites such as 3-ene VPA, 4-ene VPA, (E)- and ( Z ) - 2 , 4 - d i e n e VPA, and (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA (Abbott et al., 1986; Kassahun et al., 1990). The hepatic d i s t r i b u t i o n of 3-ene VPA and 4-ene VPA was s i m i l a r to that of 2-ene VPA, 3-OH VPA, and 3-keto VPA and hence, i t was assumed that these unsaturates were not microsomal but rather products of mitochondrial or peroxisomal metabolism (Granneman et al., 1984). However, R e t t i e et al. (1987) have shown that 4-ene VPA i s a product of microsomal VPA metabolism and that the production of 4-ene VPA i s s u s c e p t i b l e to cytochrome P-450 enzyme induction or i n h i b i t i o n . The remaining unsaturated metabolites are considered to be secondary metabolites of VPA. The o r i g i n of these secondary metabolites i s u n c l e a r , p r i m a r i l y as a r e s u l t of m u l t i p l e transformations and merging metabolic pathways. The reduction of several unsaturated metabolites of VPA has been found to o c c u r , a l b e i t to a minor extent. In r a t s , f o l l o w i n g 2-ene VPA or 3-ene VPA a d m i n i s t r a t i o n , 2% of the dose was recovered as VPA (Granneman et al., 1984). S i m i l a r r e s u l t s were seen i n r a t s f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n i n which 3-ene VPA, (E)-2-ene VPA, and VPA were noted i n the plasma (Abbott et al., 1987). Two other minor routes of VPA metabolism are the epoxidation of the unsaturated metabolites to y i e l d hydroxylactones, and g l y c i n e conjugation of which each accounts f o r <0.1% of the administered dose (Granneman et al., 1984; Rettenmeier et al., 1985; 1986). 1.1.4 Mechanism of A c t i o n The mechanism of a c t i o n of VPA i s not well understood. C u r r e n t l y there are three proposed mechanisms of a c t i o n : 1) VPA increases b r a i n GABA l e v e l s (Godin et al., 1969); 2) VPA p o t e n t i a t e s the postsynaptic response to GABA (MacDonald and Bergey, 1979); and 3) VPA exerts a d i r e c t membrane e f f e c t ( S l a t e r and Johnson, 1978). Although there i s considerable evidence to support each hypothesis the mechanism s t i l l remains u n c l e a r . Each proposed mechanism of a c t i o n of VPA w i l l be b r i e f l y discussed below. 1.1.4.1 The GABA System •y-Aminobutyric a c i d (GABA) functions as a neurotransmitter that i n h i b i t s the f i r i n g of an assortment of neurons by binding to the GABA/\ r e c e p t o r , thereby i n c r e a s i n g the permeability of postsynaptic membranes to c h l o r i d e i o n s . This causes the membrane to become h y p e r p o l a r i z e d , i n c r e a s i n g the threshold f o r the i n i t i a t i o n of an a c t i o n p o t e n t i a l ( S t r y e r , 1988). The GABA/\ receptor i s l o c a t e d predominantly i n postsynaptic membranes and i s associated with other binding s i t e s that i n c l u d e the benzodiazepine, b a r b i t u r a t e , and p i c r o t o x i n (Cotariu et al., 1990). GABA i s widely d i s t r i b u t e d throughout the b r a i n . On a c e l l u l a r l e v e l , GABA uptake i s greatest i n the nerve terminals but there i s some uptake by the neuronal c e l l bodies and g l i a l c e l l s . The s u b s t a n t i a n i g r a contains the highest amount of GABA of a l l b r a i n n u c l e i and 80% of t h i s GABA i s l o c a t e d i n the nerve terminals (Iadarola and Gale, 1982). GABA i s a simple amino a c i d synthesized from glutamate v i a the enzyme glutamic a c i d decarboxylase (GAD). Catabolism of GABA by the enzyme GABA-or-oxoglutarate aminotransferase (GABA-T) y i e l d s s u c c i n i c semialdehyde (SSA) which i s f u r t h e r o x i d i z e d to succinate and then enters the Krebs c y c l e . The pathway f o r producing and e l i m i n a t i n g GABA i s known as the GABA shunt. Modulation of the enzymes w i t h i n the GABA shunt can u l t i m a t e l y vary the amount of GABA and hence, i n f l u e n c e neuronal a c t i v i t y . For example, an i n h i b i t o r of GABA-T, 7 - v i n y l GABA, has been shown to increase brain GABA l e v e l s up to f i v e f o l d (Schecter et al., 1977). Thus an increase or p o t e n t i a t i o n of GABA a c t i v i t y i n the b r a i n by anticonvulsant agents seems to be a p l a u s i b l e mechanism of a c t i o n . 1 .1.4 .2 Increase of Brain GABA VPA has been shown to elevate GABA l e v e l s i n the whole b r a i n of rodents w i t h i n 15 - 60 minutes of a d m i n i s t r a t i o n (Schechter et al., 1978; Perry and Hansen, 1978) and these remain elevated f o r 3 - 8 hours (Schechter et al., 1978; Nau and Loscher, 1982). The increase i n GABA occurs p r i m a r i l y i n the substantia n i g r a , an area of high GABA content, although the m a j o r i t y of t h i s increase i s seen i n the nerve terminals (Iadarola and Gale, 1981). I t should be noted that an increase i n GABA l e v e l s i s only suggestive of anticonvulsant a c t i o n as researchers have yet to show that VPA d i r e c t l y r e s u l t s i n an increased r e l e a s e of GABA. The increase i n b r a i n GABA l e v e l s by VPA was thought to occur v i a the i n h i b i t i o n of GABA-T. GABA-T a c t i v i t y has been shown to decrease f o l l o w i n g VPA a d m i n i s t r a t i o n (Horton et al., 1977). However, i n h i b i t o r s of GABA-T, 7 . e . 7 - v i n y l GABA and ethanolamine-O-sulfate, were not as potent as VPA i n i n h i b i t i n g MES or PTZ induced seizures i n mice (Loscher, 1981) or r a t s (Iadarola and Gale, 1981). It has r e c e n t l y been suggested that an increase i n GABA i s a l s o associated with the e f f e c t s of VPA on s u c c i n i c semi aldehyde dehydrogenase (SSADH), the enzyme that converts s u c c i n i c semialdehyde (SSA) to succinate (Vayer et al., 1988). It was proposed that VPA i n h i b i t s SSADH thereby r e s u l t i n g i n a pool of SSA that can e i t h e r i n h i b i t GABA-T, or upon t r a n s a m i n a t i o n , be converted to GABA. E i t h e r route u l t i m a t e l y r e s u l t s i n a net increase i n GABA. Conversely, e l e v a t i o n of GAD a c t i v i t y , the GABA s y n t h e s i z i n g enzyme, has been observed i n mice f o l l o w i n g continuous a d m i n i s t r a t i o n with VPA (Loscher and Nau, 1982; Nau and Loscher, 1982 and 1984). The increase i n GAD a c t i v i t y was r e l a t e d to an increase i n b r a i n GABA l e v e l which corresponded to an e l e v a t i o n i n the e l e c t r o c o n v u l s i v e t h r e s h o l d . While these r e s u l t s suggest that the anticonvulsant a c t i v i t y of VPA i s due to e l e v a t i o n s of brain GABA, there are reports that have suggested that e l e v a t i o n of brain GABA by VPA may not be the mechanism of a c t i o n . For example, c o r r e l a t i o n between brain GABA l e v e l s and the anticonvulsant a c t i v i t y of VPA using electroshock t e s t i n g has been poor (Kerwin et al., 1980). At the lower doses of VPA used to block audiogenic or e l e c t r i c a l l y induced seizures i n mice, no changes i n t o t a l GABA l e v e l s i n the brain were observed (Kupferberg, 1980). Thus, other mechanisms of a c t i o n f o r VPA should be considered u n t i l these f i n d i n g s can be r a t i o n a l i z e d . 1.1.4.3 P o t e n t i a t i o n of Postsynaptic GABA Based on studies employing i s o l a t e d s p i n a l cord neurons, VPA has been shown to p o t e n t i a t e GABA-mediated postsynaptic i n h i b i t i o n when applied m i c r o i o n t o p h o r e t i c a l l y (MacDonald and Bergen, 1979). The p o t e n t i a t i o n of GABA by VPA was a l s o observed i n the r a t c o r t i c a l neurons i n the s u b s t a n t i a n i g r a (Kerwin et al., 1980). Although VPA-mediated postsynaptic p o t e n t i a t i o n of GABA i s a favored mechanistic e x p l a n a t i o n , i t i s not without shortcomings. The concentration of VPA i n i t i a l l y used to p o t e n t i a t e GABA response was higher than that seen in vivo (Harrison and Simmonds, 1982) and when the concentration was reduced to r e f l e c t serum l e v e l s , the r e s u l t s of MacDonald and Bergen (1979) could not be repeated. Ticku and Davis (1981) had suggested that the i n t e r a c t i o n of VPA with the GABA/\ receptor at the p i c r o t o x i n s i t e might be the mechanism by which VPA i n t e r a c t s with GABA; however, upon f u r t h e r i n v e s t i g a t i o n using a t r i t i a t e d analogue no evidence of binding to b r a i n membranes was found (Morre et al., 1984). 1 . 1 . 4 . 4 D i r e c t Membrane E f f e c t An increase i n membrane conductance to potassium by VPA has been observed i n the A p l y s i a neuron, an i n v e r t e b r a t e model; however, the concentrations used were 15 - 50 times greater than the l e v e l s seen i n p a t i e n t serum ( S l a t e r and Johnston, 1978). Incubation of hippocampal s l i c e s i n a low calcium and high magnesium bath blocked synaptic transmissions r e s u l t i n g i n rhythmic d i s c h a r g e s . These discharges can be reduced with VPA at concentrations s i m i l a r to that seen i n serum (Agopyan et al., 1986). Nevertheless, before t h i s mechanism of VPA a c t i v i t y i s to gain prominence, the involvement of potassium e f f l u x e s with e p i l e p t i c a c t i v i t y must be determined. 1 . 1 . 5 Pharmacokinetics A f t e r o r a l a d m i n i s t r a t i o n of VPA i n man, the drug i s r a p i d l y and completely absorbed with peak plasma l e v e l s a t t a i n e d w i t h i n 0 . 5 - 2 hours and the f r a c t i o n of the dose absorbed c l o s e to 1 (Schobben et al., 1975; Loiseau et al., 1975; Meinardi et al., 1975; Perruca et al., 1978). Although the absorption of VPA can be delayed a f t e r a meal, the extent of absorption i s not a f f e c t e d (Levy et al., 1980). The e l i m i n a t i o n h a l f - l i f e of VPA i n plasma ranges from 8 - 1 6 hours i n adult e p i l e p t i c s ( K l o t z and Antonin, 1977; Loscher, 1978; Bowdle et al., 1980; Gugler and von-Unruh, 1980) and 3 - 1 2 hours i n c h i l d r e n (Cloyd et al., 1983). However, p a t i e n t s on m u l t i p l e a n t i e p i l e p t i c drug therapy have s i g n i f i c a n t l y shorter h a l f - l i v e s of VPA, a r e s u l t of hepatic enzyme i n d u c t i o n (Gugler and von Unruh, 1980; Cloyd et al., 1983). The clearance of VPA i s independent of l i v e r blood flow but i s dependent on the f r e e f r a c t i o n . Thus, VPA i s a r e s t r i c t i v e l y cleared drug (Gugler and von Unruh, 1980; Levy, 1984). The clearance of VPA can vary with age and polytherapy. Children <5, 5 - 10, and 10 - 15 years of age have reported mean VPA clearances of 4 8 . 3 , 3 9 . 1 , and 24.8 mL/kg/h, r e s p e c t i v e l y (Dodson and Tasch, 1981) whereas adult clearances of VPA range from 6 - 10 mL/kg/h ( K l o t z and Antonin, 1977; Wulff et al., 1977). The clearance of VPA was found to increase (14.4 - 16.5 mL/kg/h) i n adults on polytherapy, again a r e s u l t of hepatic enzyme induction (Schappel et al., 1980; Hoffman et al., 1981). The volume of d i s t r i b u t i o n of VPA i s r e l a t i v e l y small i n both c h i l d r e n and a d u l t s . A value of 0.1 - 0.4 L/kg i s an i n d i c a t i o n that the d i s t r i b u t i o n of VPA i s l i m i t e d to the c i r c u l a t i o n and r a p i d l y exchangeable e x t r a c e l l u l a r waters (Klotz and Antonin, 1977; Gugler and von Unruh, 1980). However, the volume of d i s t r i b u t i o n of the unbound drug i n plasma i s approximately 1 L/kg, a r e f l e c t i o n of the d i s t r i b u t i o n of f r e e VPA i n t o the i n t r a c e l l u l a r s t r u c t u r e s (Gugler et al., 1977). Therapeutic plasma concentrations of VPA vary w i d e l y , although the recommended value f o r c l i n i c a l purposes i s between 50 - 100 ug/mL (Chadwick, 1985). The c o r r e l a t i o n between plasma l e v e l and dose has been poor (Loiseau et a l . , 1975; Levy, 1984). G r a p h i c a l l y , the r e l a t i o n s h i p between dose and s t e a d y - s t a t e t o t a l plasma concentration i s c u r v i l i n e a r where an increase i n the dose i s followed by a d i s p r o p o r t i o n a t e increase i n drug plasma l e v e l s . The most l i k e l y explanation f o r t h i s observation i s an increase i n free f r a c t i o n i n conjunction with the higher doses. At normal therapeutic l e v e l s VPA i s 90 - 95% bound to plasma p r o t e i n s (Bowdle et al., 1980; Gugler and von-Unruh, 1980). Plasma VPA concentrations of >100 ug/mL can r e s u l t i n a 50% increase i n f r e e drug i n plasma as a r e s u l t of the s a t u r a t i o n of VPA binding to plasma p r o t e i n s (Gugler and von-Unruh, 1980). The increase i n f r e e f r a c t i o n can cause e i t h e r an increase i n drug clearance or an increase i n the d i s t r i b u t i o n of the drug i n t o the i n t r a c e l l u l a r compartments (May and Rambeck, 1985). The u l t i m a t e r e s u l t i s a net decrease of t o t a l VPA i n plasma. The use of f r e e plasma VPA concentrations rather than t o t a l concentrations to assess the appropriateness of dose appears to be a r a t i o n a l d e c i s i o n . However, a poor c o r r e l a t i o n was a l s o found between f r e e plasma VPA concentrations and the dose required to achieve seizure c o n t r o l ( F a r r e l l et al., 1986). Therefore, the a b i l i t y to p r e d i c t appropriate therapeutic l e v e l s of VPA based on dose could be d i f f i c u l t (Armijo et al., 1986). 1 .1 .6 Pharmacodynamics The r e l a t i o n s h i p between plasma VPA concentration and pharmacological e f f e c t has a l s o been i n v e s t i g a t e d . In a study of 61 c h i l d r e n on VPA anticonvulsant mono- or polytherapy, 21 of the 24 s e i z u r e f r e e - c h i l d r e n had serum VPA concentrations between 20 - 60 ug/mL ( F a r r e l l et al., 1986). Attempts at c o r r e l a t i n g t o t a l and f r e e plasma VPA concentrations (Gram et al., 1977; Schulz et al., 1979; Bowdle et al., 1980; Cramer et al., 1986) or b r a i n (Loscher et al., 1988) and CSF (Loscher et al., 1988) l e v e l s with anticonvulsant a c t i v i t y i n both man and animals have not been s u c c e s s f u l . This discrepancy between e f f e c t and concentration e x i s t s i n - p a r t because the a n t i e p i l e p t i c e f f e c t of VPA develops independent of serum concentrations (Chadwick, 1985). Upon i n i t i a l a d m i n i s t r a t i o n of VPA, a delay was noted i n the onset of maximal anticonvulsant a c t i v i t y (Jeavons and C l a r k , 1974; Rowan et al., 1979), together with an extended duration of a c t i v i t y even a f t e r the disappearance of the parent compound from the c i r c u l a t i o n (Lockard and Levy, 1976; Harding et al., 1978). The duration of t h i s post-drug e f f e c t has been reported to l a s t f o r weeks i n both p a t i e n t s and animals (Lockard and Levy, 1976; Harding et al., 1978). Since VPA i s e x t e n s i v e l y metabolized by the l i v e r , perhaps one or more metabolites may possess s i g n i f i c a n t anticonvulsant a c t i v i t y and hence, serum concentrations of these metabolites may be a b e t t e r i n d i c a t o r of a c t i v i t y . 1.1.7 Anticonvulsant A c t i v i t y of VPA Metabolites The slow onset of maximal anticonvulsant a c t i v i t y of VPA could i n f a c t be due to the gradual accumulation of a c t i v e m e t a b o l i t e s , whereas the c a r r y - o v e r e f f e c t may be a t t r i b u t e d to the metabolite(s) possessing a longer h a l f - l i f e . Several VPA metabolites have been evaluated f o r t h e i r p o t e n t i a l to suppress e i t h e r an e l e c t r i c a l l y or chemically induced s e i z u r e i n mice (Loscher, 1981; Loscher and Nau, 1985; Abbott and Acheampong, 1988). Of the metabolites tested (E)-2-ene VPA, was shown to be 50 -100% as potent as VPA. Furthermore, upon chronic a d m i n i s t r a t i o n of VPA to mice (Nau and Loscher, 1981; Loscher and Nau, 1982) and r a t s (Loscher and Nau, 1983), (E)-2-ene VPA was the only metabolite found i n the b r a i n . In a d d i t i o n , (E)-2-ene VPA remained i n the b r a i n and p a r a l l e l e d the prolonged anticonvulsant a c t i v i t y of VPA even a f t e r the disappearance of the parent drug from the plasma. In another study, the continuous i . v . i n f u s i o n of VPA i n the dog r e s u l t e d i n i n c r e a s i n g l e v e l s of (E)-2-ene VPA i n plasma and CSF over 7 hours (Loscher and Nau, 1983). In other words, the metabolite (E)-2-ene VPA appeared to be accumulating. Upon the o r a l a d m i n i s t r a t i o n of VPA to healthy v o l u n t e e r s , at s t e a d y - s t a t e the unsaturated metabolites appeared to have longer plasma h a l f - l i v e s when compared to the parent drug ( P o l l o c k et al., 1986). These r e s u l t s were i n keeping with the idea that a slowly accumulating metabolite with a longer h a l f - l i f e may be responsible f o r the unusual pharmacodynamic behavior of VPA. On the other hand, the penetration of (E)-2-ene VPA i n t o the CSF of p a e d i a t r i c p a t i e n t s on VPA therapy was found to be l e s s than 1% of that i n plasma (Loscher et al., 1988). Although actual b r a i n l e v e l s of (E)-2-ene VPA were not determined, i t could be concluded from the low l e v e l s of (E)-2-ene VPA found i n the CSF that t h i s metabolite does not c o n t r i b u t e s i g n i f i c a n t l y to the anticonvulsant actions of VPA. A diunsaturated m e t a b o l i t e , 2 , 3 ' - d i e n e VPA, was shown to possess anticonvulsant a c t i v i t y towards PTZ-induced seizures i n mice and was found to be equipotent to (E)-2-ene VPA (Abbott and Acheampong, 1988). Although there are four p o s s i b l e geometric isomers of t h i s d i e n e , only two isomers have been i d e n t i f i e d i n man, namely the (E ,Z)- and (E ,E)-isomers (Lee et al., 1989). The ( E , E ) - 2 , 3 ' - d i e n e VPA i s considered to be a major metabolite of VPA i n man with serum l e v e l s between 6.4 - 7.1% of that of VPA (Abbott et al., 1986; Kassahun et al., 1990). 1.2 ANTICONVULSANT EVALUATION The current method of evaluating a new compound f o r i t s a n t i e p i l e p t i c p o t e n t i a l i s by the use of animal models. An i d e a l model should possess a l l of the pathophysiological components of e p i l e p s y and yet be able to assess a l a r g e number of compounds. Furthermore, the method should be able to evaluate the anticonvulsant a c t i v i t y f o r a l l types of e p i l e p s i e s and must be cost and labour e f f e c t i v e because of the l a r g e number of compounds to be screened. As expected, there i s c u r r e n t l y no such a t e s t that f i t s a l l of the aforementioned c r i t e r i a . In-vivo s t u d i e s using mice and r a t s have become an a t t r a c t i v e a l t e r n a t i v e f o r assessing anticonvulsant a c t i v i t y ( K r a l l et al., 1978). Because there i s not a s i n g l e animal model to d e s c r i b e a l l the e p i l e p s i e s seen i n humans, a number of t e s t s are performed f o r a complete assessment of the anticonvulsant a c t i v i t y of a new compound. 1.2.1 E l e c t r i c a l l y Induced Seizures Electroshock induced seizures can be categorized i n t o two types: t h r e s h o l d and maximal electroshock s e i z u r e s . A threshold induced s e i z u r e e l i c i t s an e l e c t r i c a l discharge i n the brain that occurs r e g i o n a l l y with the spread to adjacent areas kept to a minimum. Seizures of t h i s type are analogous to human absence epilepsy. Seizures e l ic i ted via maximal electroshock also stimulate neurons on a regional basis but unlike threshold induced seizures, the e lectr ical act iv i ty spreads throughout the entire brain. A seizure produced by maximal electroshock is similar to a grand mal seizure. Therefore, the appropriate test should be applied when evaluating a compound that is targeted for a specif ic form of epilepsy. Threshold seizures can be induced in mice by low frequency electroshock stimulation (LES) at 6 Hz for 3 sec via corneal electrodes (Swinyard, 1 9 7 2 ) . The application of a 2 3 V stimulus wil l stun the mouse whereas a 1 3 6 V stimulus wil l cause a seizure analogous to human absence epilepsy. A stimulation intensity > 1 5 0 V wil l result in hindleg tonic-extension akin to generalized tonic-clonic seizures. Maximum electroshock seizures (MES) are e l ic i ted with voltages greater than that required for LES (Toman et al., 1 9 4 6 ) . Corneal application of a 6 0 Hz stimulus for 0 . 2 sec at 2 5 0 V wil l e l i c i t tonic hindlimb extension (endpoint) in mice (Swinyard, 1 9 7 2 ; Swinyard and Woodhead, 1 9 8 2 ) . 1 . 2 . 2 Chemically Induced Convulsions A number of chemical convulsants are available for test ing. These include picrotoxin, b icucul l in , strychnine, and pentylenetetrazol e (PTZ). Of these, PTZ is the most commonly used chemical convulsant. Pentylenetetrazole can induce two types of seizures depending on the dose. Although the mechanism of action is not fu l ly understood, PTZ is thought to be a synaptically acting convulsant which may direct ly activate excitatory synapses (Purpura and Gonzalez-Monteagudo, 1 9 6 0 ) . P e n t y l e n e t e t r a z o l e apparently acts a l l o s t e r i c a l l y on the GABA-benzodiazepine receptor (Ramajaneyulu and T i c k u , 1984). Subcutaneous a d m i n i s t r a t i o n of 85 mg/kg of PTZ i n mice or 75 mg/kg i n r a t s produces c l o n i c seizures which are considered to be a model f o r human absence seizures (Mares and Schickerova, 1980; Swinyard and Woodhead, 1982). This type of seizure i s a l s o termed a threshold c o n v u l s i o n . The c l o n i c spasms are c h a r a c t e r i z e d i n i t i a l l y by i s o l a t e d myoclonic j e r k s w i t h i n the f i r s t 5 minutes followed by clonus of the head and f o r e l i m b areas ( V e l i s e k et a 7 . , 1989; Yonekawa et a 7 . , 1980). A Straub t a i l phenomenon and l o s s of r i g h t i n g may a l s o occur followed f i n a l l y by a stupor phase or unusual p o s t u r i n g . Doses of >100 mg/kg s . c . i n mice w i l l e l i c i t a generalized t o n i c - c l o n i c s e i z u r e , s i m i l a r to that induced by MES, and i s u s u a l l y f a t a l ( V e l i s e k et al., 1989). 1.3 OBJECTIVES The pharmacokinetic and metabolic p r o f i l e s of VPA have been e x t e n s i v e l y s t u d i e d . However, s i m i l a r work on the a c t i v e metabolites of VPA i s incomplete. The c o n t r i b u t i o n of (E)-2-ene VPA and ( E , E ) - 2 , 3 ' -diene VPA towards VPA anticonvulsant a c t i v i t y have so f a r been s p e c u l a t i v e . D e t a i l e d k i n e t i c and d i s t r i b u t i v e s t u d i e s are l a c k i n g and need to be completed before commenting on the degree of involvement of these metabolites with VPA anticonvulsant a c t i v i t y . The aim of t h i s study was to provide the pharmacokinetic data and evaluate the anticonvulsant a c t i v i t y of VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA to adequately assess the r o l e of these metabolites towards the anticonvulsant a c t i v i t y of VPA. The metabolism of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA was a l s o examined i n order to determine i f these unsaturates could p o s s i b l y replace VPA as an anticonvulsant agent. The absence of t o x i c by-products from (E)-2-ene VPA or ( E , E ) - 2 , 3 ' - d i e n e VPA metabolism would be a major advantage over VPA. 1.3.1 Spe c i f i c Aims i . To s t e r e o s e l e c t i v e l y synthesize s u f f i c i e n t amounts of both (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA f o r pharmacokinetic and pharmacological e v a l u a t i o n . i i . To develop a method f o r the a n a l y s i s of VPA and i t s metabolites i n r a t t i s s u e e x t r a c t s by negative ion chemical i o n i z a t i o n GC-MS. 28 i i i . To determine and compare the t i s s u e d i s t r i b u t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n the r a t a f t e r a s i n g l e dose. i v . To determine and compare the e l i m i n a t i o n k i n e t i c s of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n the r a t . v . To q u a n t i t a t e the metabolites formed i n the various t i s s u e f r a c t i o n s a f t e r the a d m i n i s t r a t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA. v i . To compare and quantitate the anticonvulsant a c t i v i t y of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s using the PTZ-induced seizure t e s t . 2. EXPERIMENTAL SUPPLIES . 1 Chemicals and Reagents A l d r i c h Chemical Co. (Milwaukee, WI) a - b r o m o - 2 , 3 , 4 , 5 , 6 - p e n t a f l u o r o t o u l e n e , t - b u t y l - d i m e t h y l s i l y l c h l o r i d e , b u t y l l i t h i u m (1.6 M i n hexane), deuterochloroform, l , 8 - d i a z a b i c y c l o [ 5 . 4 . 0 ] undec-7-ene (DBU), D i a z a l d R , d i c h l o r o d i m e t h y l s i l a n e , d i i s o p r o p y l a m i n e , d i i s o p r o p y l e t h y l -amine, hexamethylphosphoramide, methanesulfonyl c h l o r i d e , 2-m e t h y l g l u t a r i c a c i d , 1 ,5-pentamethylenetetrazole (pentylene-t e t r a z o l , PTZ), (E)-2-pentenoic a c i d , t r i e t h y l a m i n e BDH Chemicals (Toronto, Ont.) d i e t h y l ether (anhydrous), h y d r o c h l o r i c a c i d , q u i n o l , sodium c h l o r i d e , sodium hydroxide, sodium s u l f a t e , s u l f u r i c a c i d , tetrahydrofuran (THF), toluene Caledon Laboratories L t d . (Georgetown, Ont.) dichloromethane, ethyl acetate Eastman Kodak Co. (Rochester, NY) propionaldehyde ICN Pharmaceuticals Inc. ( P l a i n v i e w , NY) d i - n - p r o p y l a c e t i c a c i d ( v a l p r o i c acid) Manostat (New York, NY) Chromerge^ Matheson Coleman and B e l l Co. (Norwood, OH) 2-(2-ethoxyethoxy)ethanol P i e r c e (Rockford, IL) N-methyl-N-trimethylf1uoroacetamide 2 . 1 . 2 VPA Metabolites and Internal Standards The syntheses f o r the f o l l o w i n g VPA m e t a b o l i t e s , used as standards f o r q u a n t i t a t i o n , has been described elsewhere (Acheampong et al., 1983): 3-ene VPA (stereochemistry not determined), 4-ene VPA, 3-OH VPA, 4-0H VPA, 5-0H VPA, 4-keto VPA, and 2-PGA. In a d d i t i o n , ( E ) - 2 , 4 - d i e n e VPA was synthesized according to the procedure of Lee et al. (1989) w h i l e 3-keto VPA was synthesized by a l k y l a t i o n of ethyl v a l e r a t e with propionyl c h l o r i d e f o l l o w i n g the general procedure of Cregge et al. (1973). The i n t e r n a l standards used f o r the assay of VPA and VPA metabolites were [2H]eVPA, synthesized p r e v i o u s l y by Acheampong et al. (1984); [2H]32-ene VPA, synthesized by Abbott et al. (1986); [ 2 H] 3 3-keto VPA was a kind g i f t from Dr. T.A. B a i l l i e ( U n i v e r s i t y of Washington, School of Pharmacy, S e a t t l e , WA); and 2 - m e t h y l g l u t a r i c a c i d (2-MGA). 31 2 . 1 . 3 M a t e r i a l s A l l t e c h Associates Inc. ( D e e r f i e l d , IL) 1 mL amber crimp-top v i a l s , 100 uL l i m i t e d volume i n s e r t s Ami con Corp. (Danvers, MA) m i c r o p a r t i t i o n system MPS-1, u l t r a f i l t r a t i o n membrane type YMT Becton Dickinson (Rutherford, NJ) Yale hypodermic needle 23G 1, lmL syringe ( t u b e r c u l i n ) J & W S c i e n t i f i c (Folsom, CA) DB-1 c a p i l l a r y column (30m, 0.32mm i . d . , 0.25 urn f i l m t h i c k n e s s ) Restek Corp. ( B e l l e f o n t e , PA) phenyl-methyl deactivated guard column (5m, 0.32 mm i . d . ) Supelco Inc. ( B e l l e f o n t e , PA) 3% Dexsil 300 on 100-120 mesh Supelcoport, G l a s S e a l R connector 2 . 1 . 4 Animals Male Sprague-Dawley r a t s weighing between 200 - 350 g were obtained from the Animal Care F a c i l i t y at the U n i v e r s i t y of B r i t i s h Columbia. The animals were housed i n p l a s t i c cages with wood shavings used as bedding. The animals were allowed access to food (Purina Rat Chow^) and water ad libitum. Light was provided between the hours of 6:00 am to 9:00 pm d a i l y w h i l e room temperature was kept at a constant 2 2 ° C . 2.2 INSTRUMENTATION 2.2.1 Nuclear Magnetic Resonance Spectrometry High f i e l d proton NMR spectra were obtained on a Bruker WH-400 and a Varian XL-300 spectrometer i n the Department of Chemistry, U n i v e r s i t y of B r i t i s h Columbia NMR f a c i l i t y . Spectra were acquired i n deuterated chloroform with t e t r a m e t h y l s i l a n e used as an i n t e r n a l standard. 2.2.2 Packed Column Gas Chromatography - Mass Spectrometry The i d e n t i f i c a t i o n of synthesized compounds was performed on a Hewlett Packard 5700A gas chromatograph i n t e r f a c e d to a Varian MAT-111 mass spectrometer equipped with a v a r i a b l e s l i t separator. Data was acquired and processed on a microcomputer. Data were recorded i n scan mode with a mass range of 15 - 750 mass u n i t s c o l l e c t e d every 5 seconds. The instrument was operated i n e l e c t r o n impact mode with an emission current of 300 uA, i o n i z a t i o n energy of 70 eV, and source pressure of 5 x 10"6 T o r r . The column (1.8 m x 2 mm i . d . ) was packed with 3% Dexsil 300 on 100 - 120 mesh Supelcoport. The oven temperature program used was 50 -300°C at 16°C/min. I n j e c t i o n port temperature was 250°C and the separator temperature set at 250°C. The c a r r i e r gas, with a flow of 25 mL/min, was helium. 2 . 2 . 3 C a p i l l a r y Column Gas Chromatography - Mass Spectrometry The q u a n t i t a t i v e a n a l y s i s of b i o l o g i c a l samples f o r VPA and metabolites was performed on a Hewlett Packard 5987A GCMS with an open-s p l i t i n t e r f a c e . Data recording and processing were managed with a HP-1000 o n - l i n e computer. Negative ion chemical i o n i z a t i o n (NICI) was the method of i o n i z a t i o n with u l t r a - h i g h p u r i t y methane used as the reagent gas. The source pressure was 0.8 - 1.2 T o r r , i o n i z a t i o n energy 200 eV, and the emission current 250 uA. The instrument was programmed f o r s e l e c t e d - i o n - m o n i t o r i n g (SIM) to enhance s e n s i t i v i t y . VPA and metabolites assayed and the ions monitored are l i s t e d i n t a b l e 3 . Separation was achieved on a DB-1 f u s e d - s i l i c a column connected to a phenyl-methyl deactivated 5 m guard column by way of a GlasSeal^ connector. The c a r r i e r gas used was helium with a head pressure of 10 psi and a r e s u l t i n g flow rate of 1 mL/min. The oven temperature program was 110 - 140°C at 30°C/min, then 140 - 260°C at 8 ° C / m i n , and f i n a l l y held at 280°C f o r 6 min f o r a t o t a l run time of 25 min. A 1 uL a l i q u o t of sample was i n j e c t e d using a Hewlett Packard 7673A automatic sampler. 2 . 2 . 4 Centrifuges A Beckman c e n t r i f u g e , model J2-21 , equipped with a JA-20 45° r o t o r was used to separate the unbound drug from the plasma by u l t r a f i l t r a t i o n . A Damon/IEC c e n t r i f u g e , model HN-SII f i t t e d with a 45° r o t o r , was used to separate the organic and aqueous l a y e r s a f t e r Table 3 : Mass-to-charge r a t i o s (m/z) f o r the i n t e r n a l standards VPA, and VPA m e t a b o l i t e s . COMPOUND m/z [ 2 H 3 ]2-ene VPA* 144 [2H6]VPA* 149 [ 2 H 3 ]3-keto VPA* 232 2-MGA* 325 ( E , E ) - 2 , 3 ' - d i e n e VPA 139 ( E , Z ) - 2 , 3 ' - d i e n e VPA 139 ( E ) - 2 , 4 - d i e n e VPA 139 (E)-2-ene VPA 141 (Z)-2-ene VPA 141 3-ene VPA 141 4-ene VPA 141 VPA 143 p4-keto VPA 157 3-keto VPA 229 3-OH VPA 231 4-0H VPA 231 5-0H VPA 231 2-PGA 353 e x t r a c t i o n by gentle mechanical r o t a t i o n of the t i s s u e homogenates or plasma samples. 2.3 CHEMICAL SYNTHESES 2 . 3 . 1 Synthesis of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d ((E)-2-ene VPA) 2 . 3 . 1 . 1 Synthesis of ethyl 2-n-propyl-3-hydroxypentanoate In a 500 mL f l a s k equipped with a dropping f u n n e l , r e f l u x condenser, and mechanical s t i r r e r , n - b u t y l l i t h i u m i n hexane (0.025 mol, 156 mL of 1.6M i n hexane) was added to a cooled s o l u t i o n of diisopropylamine (0.25 mol, 25.3g) i n 200 mL of THF over i c e . The mixture was s t i r r e d f o r 30 minutes and cooled to - 7 8 ° C with a dry ice/acetone bath. Ethyl v a l e r a t e (0.25 mol, 32.5g) i n 10 mL THF was added dropwise over 10 minutes to the mixture and s t i r r e d a f u r t h e r 60 minutes. Upon quenching with 15% HC1 to a pH of 1, the aqueous l a y e r was extracted with 3 x 100 mL of ether and the ethereal p o r t i o n washed c o n s e c u t i v e l y with saturated NaHCC>3 and water. The ethereal s o l u t i o n was d r i e d over anhydrous Na2S04 and the ether removed by f l a s h evaporation. P u r i f i c a t i o n of the residue by f r a c t i o n a l d i s t i l l a t i o n afforded 22.8 g (48% y i e l d ) of ethyl 2-n-propyl-3-hydroxypentanoate, bp 94 - 96°C/2.5mm ( l i t . 70-72°C/0.2mm (Acheampong, 1982)). Mass spectrum (MW=188) m/z(%): 101(100), 73(67), 55(50), 113(50), 130(27), 84(20), 159(20), 143(15). 2 .3.1.2 Synthesis of 2-n-propyl-(E)-2-pentenoic a c i d In a 250 mL f l a s k equipped with a dropping funnel and mechanical s t i r r e r , ethyl 2-n-propyl-3-hydroxypentanoate (48 mmol, 9 g ) , t r i ethyl amine (72 mmol, 10 mL), and 150 mL of dichloromethane were cooled to 0"C with i c e . Methanesulfonyl c h l o r i d e (50 mmol, 4 mL) i n 10 mL of dichloromethane was cooled to 0°C and added dropwise to the s t i r r e d mixture at room temperature. A f t e r 60 min the mixture was f i l t e r e d by s u c t i o n and the solvent removed by f l a s h evaporation. The residue was r e c o n s t i t u t e d i n 150 mL of dry THF, a s o l u t i o n of 1 ,8-d i a z a b i c y c l o [ 5 . 4 . 0 . ] u n d e c - 7 - e n e (DBU, 48 mmol, 7 mL) i n 10 mL of THF was added, and the contents gently r e f l u x e d f o r 2 h over an o i l bath. The r e a c t i o n was cooled to room temperature and quenched with 100 mL of d i s t i l l e d water. The aqueous f r a c t i o n was extracted with 3 x 150 mL of ether and the combined organic portions washed c o n s e c u t i v e l y with 100 mL of 1M HCl and 1M NaOH. The ethereal f r a c t i o n was d r i e d over anhydrous Na2S04 and the solvent removed by f l a s h evaporation. To the r e s i d u e , 15 mL of 3N NaOH and 30 mL of d i s t i l l e d water were added and s t i r r e d f o r 4 days at 60°C. The mixture was cooled and e x t r a c t e d with 50 mL of ether ( d i s c a r d ) . The aqueous l a y e r was adjusted to a pH of 1 - 2 with 3N HCl and extracted with 3 x 100 mL of e t h e r . The combined ethereal l a y e r s were d r i e d over anhydrous Na2S04, the ether was removed by f l a s h evaporation, and the residue d i s t i l l e d . The c l e a r d i s t i l l a t e , bp 1204C/3mm ( l i t . 96-99°C/l.3mm (Acheampong et a / . , 1983)) y i e l d e d 2.45 g (36% y i e l d ) of 2-n-propyl-2-pentenoic a c i d . Upon methylation with diazomethane ( L e v i t t , 1973), two isomers (95% E and 5% Z) were detected by GCMS and confirmed by NMR. Mass spectrum of the (E)-isomer (as the methyl e s t e r a f t e r d e r i v a t i z i n g with diazomethane) m/z(%): 55(100), 95(83), 127(70), 156(M + ,42), 67(37), 113(10), 141(3). Mass spectrum of the (Z)-isomer (methyl e s t e r , m/z(%)): 55(100), 95(100), 127(60), 67(45), 156(M + ,25), 113(8), 141(2). 400 MHz proton NMR (CDC13): 0 .85(t ,3H,CH3-CH 2 -CH 2 ) , 1 .0(t ,3H,CH3-CH2-CH), 1.30-1.44(m,2H,CH 3 -CH 2 -CH 2 ) , 2.1-2.25(m,2H,CH 3 -CH 2 -CH 2 ) , 2 .1-2.25(m,2H,CH 3 -CH 2 -CH,E), 2.4-2.49(m,2H,CH 3 -CH 2 -CH,Z) , 5.95(t ,1H,CH=,Z) , 6.85(t,lH,CH=,E)(Comparable to l i t . values of Acheampong et al., 1983). 2.3.2 Synthesis of 2-((Z)-l' - p r o p e n y l ) - ( E )-2 - p e n t e n o i c a c i d ( ( E , Z ) -2 , 3 ' - d i e n e VPA) 2 .3 .2 .1 Synthesis of ethyl 2-(l ' - h y d r o x y p r o p y l ) - ( Z ) - 3 - p e n t e n o a t e In a 1 L f l a s k equipped with a Dean-Stark apparatus, (E)-2-pentenoic a c i d (1.25 mol, 125 g ) , ethanol (3.75 mol, 219 ml_), and concentrated H2S04 (1.25 mL) i n 500 mL of benzene were r e f l u x e d f o r 48 h. The mixture was cooled to room temperature, washed with a 200 mL s o l u t i o n of saturated NaHC03, and the organic p o r t i o n was d r i e d over MgS04. F r a c t i o n a t i o n afforded lOOg (63% y i e l d ) of ethyl (E)-2-pentenoate, bp 159°C/760mm. Mass spectrum m/z(%): 55(100), 29(70), 128(M + ,10), 83(10), 69(6) , 100(5). 300 MHz proton NMR (CDC13): 1.09(t,3H,CH 3 -CH 2 -CH=), 1 . 3 ( t , 3 H , 0 C H 2 -CH 3 ) , 2.18-2.29(m,2H,CH3-CH2-CH=), 4 .18(q,2H,0CH 2 ) , 5.82(d,1H,CH=CH, J=14Hz), 7.02(m,2H,CH3-CH2-CH=). In a 500 mL f l a s k equipped with a dropping f u n n e l , condenser, and mechanical s t i r r e r , n - b u t y l l i t h i u m (0.10 mol, 123 mL of 1.6M i n hexane) was slowly added to a cooled s o l u t i o n of diisopropylamine (0.19 mol, 26.6 mL) i n 150 mL of THF over i c e . The mixture was allowed to s t i r f o r 15 min and then cooled to - 7 8 ° C with a d r y - i c e / a c e t o n e bath. A s o l u t i o n of hexamethylphosphoramide (0.19 mol, 33.1 mL) i n 30 mL of THF was slowly introduced and a f t e r 15 min a s o l u t i o n of ethyl (E)-2-pentenoate (0.173 mol, 22.1 g) i n 30 mL of THF was added dropwise to the r e a c t i o n and s t i r r e d a f u r t h e r 30 min. Propionaldehyde (0.173 mol, 12.5 mL) i n 25 mL of THF was added dropwise to the s t i r r e d mixture and allowed to react f o r 60 min. The mixture was quenched with 15% HC1 to a pH of 1 and the aqueous l a y e r extracted with 3 x 150 mL of e t h e r . The combined organic f r a c t i o n s were c o n s e c u t i v e l y washed with a saturated NaHCC>3 s o l u t i o n and water and d r i e d over anhydrous Na2SG"4. The solvent was removed by f l a s h evaporation and the residue f r a c t i o n a t e d to a f f o r d 25.83 g (80% y i e l d ) of ethyl 2 - ( l ' - h y d r o x y l p r o p y l ) - ( Z ) - 3 - p e n t e n o a t e , bp 68 - 7 0 T / 0 . 1 mm ( l i t . 85-90°C/0.25mm (Acheampong and Abbott, 1985)). Mass spectrum (MW=186) m/z(%): 29(100), 55(87), 100(68), 128(70), 82(53), 113(4), 141(2), 157(2), 169(2). 400 MHz proton NMR (CDCI3): 0.9(t ,3H,CH 3 -CH 2 -CH0H), 1 . 2 5 ( t , 3 H , 0 C H 2 - C H 3 ) , 1.4-1.51(m,2H,CH2-CH0H), 1 .7(d,3H,CH 3 -CH), 2.8 (broad s , l H , 0 H ) , 3.42(m,1H,CH-C=0), 3 . 8 (m,lH,CH-0), 4.15(q,2H,0CH 2 CH 3 ) , 5.3-5.61(m,lH,CH=CH, J=9Hz), 5.62-5.85(m,1H,CH=CH)(1 i t . Acheampong and Abbott, 1985). 2.3.2.2 Synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E)-2 - p e n t e n o i c a c i d In a 500 mL f l a s k equipped with a dropping funnel and s t i r r e r ethyl 2 - ( l ' - h y d r o x y p r o p y l ) - ( Z ) - 3 - p e n t e n o a t e (118 mmol, 22g) and t r i e t h y l a m i n e (177 mmol, 26 mL) i n 200 mL of dichloromethane were cooled over i c e . A cooled s o l u t i o n of methanesulfonyl c h l o r i d e (124 mmol, 10 mL) i n 10 mL of dichloromethane was then slowly added to the mixture. The i c e was removed and the r e a c t i o n was allowed to proceed at room temperature f o r 60 min. The mixture was f i l t e r e d by s u c t i o n , the solvent removed by f l a s h evaporation and the residue r e c o n s t i t u t e d i n 150 mL of THF. A s o l u t i o n of l , 8 - d i a z a b i c y c l o [ 5 . 4 . 0 ] u n d e c - 7 - e n e (118 mmol, 18 mL) i n 20 mL of THF was added and the r e a c t i o n was g e n t l y r e f l u x e d f o r 2 h. A f t e r c o o l i n g , the mixture was quenched with 100 mL of d i s t i l l e d water and the aqueous l a y e r extracted with 3 x 100 mL of e t h e r . The combined organic f r a c t i o n s were washed c o n s e c u t i v e l y with 100 mL of 1M HC1 and 1M NaOH and d r i e d over anhydrous Na2S04. The solvent was removed by f l a s h evaporation, 40 mL of 3N NaOH and 20 mL of d i s t i l l e d water were added to the residue and the mixture was s t i r r e d f o r 48 h at 60°C. Upon c o o l i n g the aqueous mixture was extracted with 100 mL of ether ( d i s c a r d ) , the pH adjusted to 1 - 2 with 3N HC1 and then extracted with 3 x 100 mL of e t h e r . The combined ethereal l a y e r was d r i e d over anhydrous Na2S04 and the solvent was removed by f l a s h evaporation. The residue was d i s t i l l e d to a f f o r d 9 . 6 g (85% y i e l d , bp 8 0 ° C / 0 . 1 mm) of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) -2-pentenoic a c i d and 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d with an isomeric r a t i o of 20:1 as determined by GC-MS and NMR. Mass spectrum (as the methyl e s t e r a f t e r d e r i v a t i z i n g with diazomethane), m/z(%): 95(100), 154(M+, 87) , 79(82), 59(58), 122(58), 67(48), 107(20), 139(20). 400 MHz proton NMR (CDC13): 1 . 0 8 ( t , 3 H , C H 3 - C H 2 ) , 1 .59(d,3H,CH 3 -CH), 2 .15(dq,2H,CH 2 ) , 5.75-5.83(m,1H,CH=CH,), 5.98(d,1H,CH=CH,J=llHz), 6.95 (t,lH,CH=C=0,J=8Hz). 2.3.3 Synthesis of 2-( ( E)- l ' - p r o p e n y l ) - ( E )-2 - p e n t e n o i c a c i d ( ( E , E ) -2,3 ' -diene VPA) D e t a i l s of the synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d are recorded here and were r e c e n t l y published (Lee et al., 1989). 2.3.3.1 Synthesis of e t h y l (Z)-2-pentenoic a c i d (Z)-2-Pentenoic a c i d was synthesized by the Favorsky rearrangement of l ,3-dibromo-2-pentenone described b r i e f l y by Rappe and Adestrom (1965) and i n d e t a i l by Rappe (1979). The ethyl (Z)-2-pentenoate was obtained by r e f l u x i n g (Z)-2-pentenoic a c i d (23.3 g , 0.23 mol) with ethyl i o d i d e (71.76 g , 0.46 mol) , potassium carbonate (23.84 g , 0.17 mol) , and 18-crown-6 (3 g , 0.05 M) i n 230 mL of dry THF f o r 6 h. The mixture was f i l t e r e d by s u c t i o n and f r a c t i o n a l d i s t i l l a t i o n afforded 17.1 g of ethyl (Z)-2-pentenoate ( y i e l d 58%, bp 43 - 45°C/10 mm). Mass spectrum m/z(%): 100(100), 83(95), 55(77), 29(65), 128(M + ,30). 80 MHz proton NMR (CDC13): l . l ( t , 3 H , C H 3 - ) , 1 . 3 ( t , 3 H , - C H 3 ) , 2.65(q,2H,CH2), 4 .2(q,2H,OCH 2 ) , 5 . 7 ( d , 1H,J=10Hz,CH=CH), 6 . 2 -6.5(dt , lH,J c i s =10Hz,J g e m =6Hz,HC=CH). 2.3.3.2 Synthesis of ethyl 2-(l ' -hydroxypropyl)-(E)-3-pentenoate In a 250 mL f l a s k equipped with a dropping f u n n e l , condenser, and mechanical s t i r r e r , diisopropylamine (11.13 g , 0.11 mol) i n anhydrous THF (90 mL) were cooled to 0°C over i c e . n - B u t y l l i t h i u m (69 mL of 1.6 M i n hexane, 0.11 mol) was then added dropwise over 15 min and allowed to react f o r 20 min. Upon c o o l i n g to -78"C, hexamethylphosphoramide (19.57 g , 0.11 mol) was added dropwise and the mixture was s t i r r e d f o r 15 min. An a l i q u o t of ethyl (Z)-2-pentenoate (12.8 g . 0.1 mol) i n 10 mL of THF was added dropwise. A f t e r 30 min, propionaldehyde (5.8 g , 0.1 mol) i n 10 mL of THF was slowly introduced and the mixture was s t i r r e d f o r 30 min. The r e a c t i o n was quenched with 15% HC1 u n t i l a pH of 1 was a t t a i n e d . The aqueous p o r t i o n was extracted three times with ether and the combined organic f r a c t i o n s washed c o n s e c u t i v e l y with saturated NaHC03 and water, then d r i e d over anhydrous Na2S04. Removal of the solvent by f l a s h evaporation and f r a c t i o n a l d i s t i l l a t i o n of the residue afforded 11.2 g ( y i e l d 60%) of ethyl 2 - ( l ' - h y d r o x y p r o p y l ) - ( E ) - 3 - p e n t e n o a t e (bp 7 8 ° C / 0 . 2 2 mm)(Lit. 95-100°C/lmm (Acheampong and Abbott (1985)). Mass spectrum (MW=186) m/z(%): 29(100), 100(50), 55(49), 82(44), 128(40), 113(5), 141(2), 157(2). 300 MHz proton NMR (CDC13): 0 .95(t ,3H,CH3-CH 2 ) , 1 .3(t ,3H,OCH 2 -CH 3 ) , 1 .45(m,2H,CH 2 ), 1.7(d,3H,CH3-CH=), 2.65(broad S , 1H,0H) , 3 . 0 ( m , l H , CH-C=0), 3 . 6 5 - 3 . 8 ( m , l H , C H - 0 ) , 4.15(q,2H,0CH 2 -CH 3 ) , 5.47(m,1H,CH=CH, J=15Hz), 5.63 (m,lH,=CH-CH). 2 . 3 . 3 . 3 Synthesis of ethyl ( E ) - 2 - ( l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o a t e In a 150 mL f l a s k equipped with a dropping funnel and mechanical s t i r r e r , ethyl 2 - ( l ' - h y d r o x y p r o p y l ) - ( E ) - 3 - p e n t e n o a t e ( 9 . 3 g , 0.05 mol) was added to a s o l u t i o n of t r i e t h y l a m i n e (8.1 g , 0.08 mol) i n 40 mL of dry dichloromethane and the mixture cooled to 0 ° C . Methanesulfonyl c h l o r i d e (6.87 g , 0.06 mol) at 0°C was slowly added and the mixture was s t i r r e d f o r 60 min. The mixture was f i l t e r e d by s u c t i o n , the solvent removed by f l a s h evaporation, and the crude mesyl e s t e r , ethyl 2 - ( l ' -hydroxypropyl)-(E)-3-pentenoate, analyzed by GCMS. Two peaks corresponding to the diastereomers gave i d e n t i c a l mass s p e c t r a . GCMS (EI) : m/z{%) 95(100), 67(55), 55(45), 123(30), 111(15), 139(15), 168(10), 153(5). The crude mixture of ethyl 2 - ( l ' - h y d r o x y p r o p y l ) - ( E ) - 3 -pentenoate was r e c o n s t i t u t e d i n 150 mL of anhydrous THF, cooled to 0 ° C , and potassium hydride (4.01 g , 0.1 mol) c a u t i o u s l y added. The mixture was then brought to 25°C and allowed to react f o r 12 h. Excess potassium hydride was c a r e f u l l y decomposed at - 7 8 ° C with water and the aqueous l a y e r extracted three times with e t h e r . The combined organic f r a c t i o n s were c o n s e c u t i v e l y washed with saturated NaHC03 and water, then d r i e d over anhydrous Na2S04. The solvent was removed by f l a s h evaporation and f r a c t i o n a l d i s t i l l a t i o n of the residue afforded 3 . 9 g ( y i e l d 46%, bp 7 0 ° C / 0 . 1 mm)(Lit. 6 5 - 7 0 ° C / 0 . 1 mm (Acheampong and Abbott, 1985)) of an isomeric mixture that by GCMS was estimated to contain 81% of ethyl 2-( ( E)- l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o a t e and 19% of ethyl 2 - ( ( Z ) - l ' - p r o p e n y l ) -(E)-2-pentenoate. Mass spectrum of ethyl 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o a t e , m/z(%): 95(100), 168(M + ,90), 79(64), 67(49), 123(46), 140(37), 111(30), 153(7). 400 MHz proton NMR (CDC13): 1.0-1.1 (t,3H,CH.3-CH2), 1 .32(t,3H,0CH2-CH 3 ) , 1.84(d,J=7Hz,3H,CH3-CH=), 2.3(m,2H,CH2-CH=), 4 . 2 ( q , 2H,0CH 2 -CH 3 ), 5.75(trace,m,CH=CH,(E,Z)) , 6.07(m,1H,CH=CH,(E,E)), 6.13 (d,J=17Hz,IH, CH=CH,(E,E)), 6 .55(t ,J=7Hz,lH,CH 2 -CH=,(E ,E)) , 6 .8(trace,CH 2 -CH=,(E ,Z)) ( L i t . Acheampong and Abbott, 1985). 2 . 3 . 3 . 4 Synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d The f r e e acids were obtained by s t i r r i n g the mixture, ethyl 2-( ( E)- l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o a t e and ethyl 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoate, (2.7 g , 0.016 mol) i n 25 mL of 2.2 M NaOH at 608C f o r 48 h. The mixture was extracted with 25 mL of hexane (discard) and the aqueous f r a c t i o n adjusted to a pH of 1 with 10% HCl. The a c i d i c s o l u t i o n was extracted three times with ether and the combined ethereal p o r t i o n s d r i e d over anhydrous Na2S04. Removal of the ether by f l a s h evaporation and f r a c t i o n a l d i s t i l l a t i o n afforded 1.2 g ( y i e l d 52.1%, bp 84°C/0.05 mm) of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d and 2 - ( ( Z ) - l ' - p r o p e n y l ) -(E)-2-pentenoic a c i d with the (E,E)-isomer c o n s t i t u t i n g approximately 94% of the m i x t u r e . Mass spectrum of the t-BDMS e s t e r d e r i v a t i v e of 2 - ( ( E ) - 1 ' -p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d , m/z(%): 197(100), 75(35), 95(20), 123(10), 254(2), 139(2), 153(2), 167(2) ( L i t . Abbott et al., 1986). Mass spectrum of the t-BDMS e s t e r d e r i v a t i v e of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d , m/z(%): 197(100), 75(20), 95(15), 123(10), 254(5), 139(2), 153(2), 167(2). 400 MHz proton NMR (CDCI3): 1 .09(t,J=8Hz,3H,CH 3 -CH2), 1 .57(trace,d,CH3-CH=,(E ,Z)) , 1.85(d,J=7Hz,3H,CH3-CH=,(E,E)), 2 . 1 1 ( t r a c e , m , C H 2 , ( E , Z ) ) , 2 . 3 4 ( m , 2 H , C H 2 , ( E , E ) ) , 5.83(trace,m,CH=CH,(E,Z)) , 5.99 ( t r a c e , d,J=HHz,CH=CH, ( E , Z ) ) , 6 . 0 2 - 6 . l l ( m , 1H,CH=CH, ( E , E ) ) , 6 . 1 5 ( d , J=16Hz,lH,CH=CH,(E,E)), 6.8(t ,J=8Hz,lH,CH 2 -CH=,(E,E)) ( L i t . Acheampong and Abbott, 1985). 2.4 ANIMAL EXPERIMENTS 2 . 4 . 1 Pharmacokinetics and Tissue D i s t r i b u t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA Aqueous s o l u t i o n s of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA f o r i . p . i n j e c t i o n were prepared as t h e i r sodium s a l t s . The concentrations used f o r VPA, (E)-2-ene VPA (corrected f o r the 5% (Z)-isomer c o n t e n t ) , and ( E , E ) - 2 , 3 ' - d i e n e VPA (corrected f o r the 3-5% (E ,Z)-isomer content) were 75, 76, and 78 mg/mL, r e s p e c t i v e l y . Each s o l u t i o n was prepared by the a d d i t i o n of an equivalent amount of 3N NaOH to a s p e c i f i e d amount of each compound i n d i s t i l l e d water to y i e l d the sodium s a l t . The pH was then adjusted to 7.4 with 3N HCl i f necessary and the mixture was made to a volume of 50 mL with d i s t i l l e d water. Male Sprague-Dawley r a t s weighing between 250 - 350g were administered 150 mg/kg i . p of VPA and s e r i a l l y s a c r i f i c e d at -15, 15, 30, 45, 60, 120, 240, 360, and 600 min with each time point c o n s i s t i n g of e i g h t r a t s . Before s a c r i f i c e , each r a t was rendered unconscious by p l a c i n g the animal i n a carbon dioxide chamber f o r 30 seconds. The animal was then decapitated with a Harvard small animal d e c a p i t a t o r . Blood was immediately c o l l e c t e d i n a heparinized t e s t tube and l a t e r c e n t r i f u g e d to recover the plasma. The l i v e r , kidneys, h e a r t , and lung of each animal were q u i c k l y removed. With the aide of a d i s s e c t i n g microscope the b r a i n was d i s s e c t e d by the use of a water a s p i r a t e d m i c r o p i p e t t e and a small s p a t u l a . The brain regions c o l l e c t e d were the hippocampus, s u p e r i o r c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , cerebellum, corpus c a l l o s u m , o l f a c t o r y b u l b s , substantia n i g r a , medulla, and caudate putamen. The remaining r a t b r a i n was also kept f o r a n a l y s i s . The l i v e r , kidneys, h e a r t , lung and remaining brain were then weighed, frozen i n l i q u i d n i t r o g e n , and stored at -80°C u n t i l assayed. Each b r a i n s e c t i o n was pooled at each time p o i n t , weighed, and stored at - 8 0 ° C . The plasma samples c o l l e c t e d were a l s o kept at - 8 0 ° C . The same experiment using the protocol above was used to obtain plasma and t i s s u e samples f o r (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. 2 . 4 . 2 Anticonvulsant Evaluation Aqueous s o l u t i o n s of VPA f o r i . p . i n j e c t i o n were prepared at concentrations of 15, 3 7 . 5 , 75, and 150 mg/mL, corresponding to doses administered of 30, 75, 150, and 300 mg/kg r e s p e c t i v e l y . For ( E , E ) - 2 , 3 ' -diene VPA s o l u t i o n s of 3 7 . 5 , 75, 100, 150, and 200 mg/mL were prepared f o r dosages administered at 75, 150, 200, 300, and 400 mg/kg, r e s p e c t i v e l y . S o l u t i o n s of 3 7 . 5 , 75, 100, and 150 mg/mL of both (E)-2-ene VPA and ( E , Z ) - 2 , 3 ' - d i e n e VPA were each prepared f o r anticonvulsant e v a l u a t i o n at dosages of 75, 150, 200, and 300 mg/kg, r e s p e c t i v e l y . S o l u t i o n s of each compound were prepared as t h e i r sodium s a l t s by the a d d i t i o n of an equivalent amount of 3N NaOH. The pH was then adjusted to 7.4 i f required with 3N HCl and the mixture made to volume with d i s t i l l e d water. An aqueous s o l u t i o n of the convulsant p e n t y l e n e t e t r a z o l e (PTZ, 3 . 5 g) was prepared d a i l y by d i s s o l v i n g the compound i n 100 mL of normal s a l i n e to y i e l d a concentration of 35 mg/mL f o r s . c . i n j e c t i o n . Male Sprague-Dawley r a t s weighing between 200 - 250 g were administered s o l u t i o n s of e i t h e r VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, or ( E , Z ) - 2 , 3 ' - d i e n e VPA i . p . at the various dosages prepared with each dose regimen r e q u i r i n g eight r a t s . Upon i n j e c t i o n , each r a t was i s o l a t e d by p l a c i n g i t i n t o c l e a r p l a s t i c cages f o r o b s e r v a t i o n . At the approximate peak b r a i n concentration f o r each compound, the convulsant PTZ was administered s . c . at a dose of 70 mg/kg. Each r a t was observed f o r a f u r t h e r 30 minutes f o r signs of seizure a c t i v i t y . The dose of PTZ administered was s u f f i c i e n t to e l i c i t a minimum s e i z u r e a l s o known as a c l o n i c s e i z u r e . The s e i z u r e i s f i r s t c h a r a c t e r i z e d by myoclonic j e r k s followed by a Straub t a i l phenomenon and then a c l o n i c c o n v u l s i o n . A c l o n i c convulsion i n r a t s i s depicted by whole body spasms followed by the arching of the spine and f l e x i n g of the jaw muscles. A paddling or swimming motion of the f r o n t paws i s h i g h l y c h a r a c t e r i s t i c of a c l o n i c convulsion with l o s s of r i g h t i n g sometimes o c c u r r i n g . A f t e r the c o n v u l s i o n s , which l a s t approximately 5 s e c , the r a t then enters a stupor or calm phase that can l a s t f o r up to one hour. A c o n t r o l group of eight r a t s was administered PTZ s . c . i n order to c h a r a c t e r i z e the s e i z u r e without the i n t e r f e r e n c e of the a n t i c o n v u l s a n t s and to determine the e f f i c a c y of the convulsant. 2.4 .3 P r o t e i n Binding of (E ,E) and (E , Z ) -2 ,3 ' - d i e n e VPA The plasma p r o t e i n binding of (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA was determined from the r a t plasma samples c o l l e c t e d during the t i s s u e d i s t r i b u t i o n and k i n e t i c s t u d i e s . The t o t a l drug bound was determined by d i r e c t l y assaying the plasma samples f o r the dienes while the f r e e drug was i s o l a t e d by the method of u l t r a f i l t r a t i o n . To obtain f r e e drug concentration from t o t a l drug i n plasma, a 500 uL a l i q u o t of plasma was placed i n t o upper chamber of an AmiconR m i c r o p a r t i t i o n apparatus f i t t e d with a YMT membrane. The e n t i r e m i c r o p a r t i t i o n apparatus was placed i n t o a Beckman c e n t r i f u g e equipped with a 45° r o t o r and spun at 1650 g f o r 20 min at 25°C. A 250 uL a l i q u o t of the u l t r a f i U r a t e was then assayed to determine the f r e e drug c o n c e n t r a t i o n . 2.5 ANALYTICAL 2.5.1 Homogenization of the Tissue Samples A l l t i s s u e samples c o l l e c t e d (heart , l u n g , b r a i n , l i v e r , and kidneys) were homogenized i n d i s t i l l e d water to uniformity and made to a s p e c i f i e d volume or weight. The remaining b r a i n , l i v e r , and kidney samples were homogenized by three passes using a Potter-Elvehjem t i s s u e g r i n d e r equipped with a Teflon p e s t l e with each t i s s u e made to volumes of 4 , 35, and 6 mL, r e s p e c t i v e l y . The heart and lung samples were homogenized f o r 20 sec over i c e using a Polytron^ equipped with a l a r g e c e l l d i s r u p t e r and made to volumes of 4 and 5 mL, r e s p e c t i v e l y . Since the sample s i z e of each d i s s e c t e d brain s e c t i o n can be very small even a f t e r p o o l i n g , a l l measurements were determined by weight to minimize e r r o r s . The weight of d i s t i l l e d water added (0.2 - 1 g) depended on the weight of each b r a i n section sample as the f i n a l concentration of brain t i s s u e must be approximately 500 mg/g. Each of the pooled b r a i n s e c t i o n samples were homogenized with water f o r 10 sec over i c e using a P o l y t r o n ^ f i t t e d with a small c e l l d i s r u p t e r . Therefore, from the weight of b r a i n s e c t i o n sample and the t o t a l weight of the homogenate, the concentration of brain material i n s o l u t i o n can be a c c u r a t e l y c a l c u l a t e d . Control t i s s u e s and plasma f o r the preparation of c a l i b r a t i o n curves were obtained from naive r a t s . The t i s s u e s were homogenized using the above procedures. The e n t i r e i n t a c t b r a i n was homogenized and used as c o n t r o l b r a i n t i s s u e f o r the a n a l y s i s of a l l b r a i n components. 2 . 5 . 2 Internal Standards A 100 mL i n t e r n a l standard s o l u t i o n was prepared by combining i n d i v i d u a l s o l u t i o n s of each compound i n t o d i s t i l l e d water to y i e l d f i n a l concentrations of 50 ug/mL f o r [2H]6VPA and 25 ug/mL f o r [2H]32-ene VPA, [ 2 H]33-keto VPA, and 2-MGA. The i n t e r n a l standard [2H]eVPA was used to q u a n t i t a t e VPA; [2H]32-ene VPA was used to q u a n t i t a t e (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA, ( E ) - 2 , 4 - d i e n e VPA, (E)- and (Z)-2-ene VPA, 3-ene VPA, 4-ene VPA, and 4-keto VPA; [2H]33-keto VPA was used to q u a n t i t a t e 3-keto VPA; and 2-MGA was used to quantitate 3-OH VPA, 4-OH VPA, 5-0H VPA, and 2-PGA. 2 . 5 . 3 C a l i b r a t i o n Curves C a l i b r a t i o n curves over a high concentration range were prepared f o r VPA, (E)- and (Z)-2-ene VPA, and (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA. C a l i b r a t i o n curves over a low concentration range were prepared f o r VPA, (E) and (Z)-2-ene VPA, and (E,E) and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n a d d i t i o n to ( E ) - 2 , 4 - d i e n e VPA, 3-ene VPA, 4-ene VPA, 4-keto VPA, 3-keto VPA, 3-OH VPA, 4-OH VPA, 5-0H VPA, and 2-PGA. For the preparation of the high concentration range c a l i b r a t i o n c u r v e s , approximately 50 mg of VPA, (E)-2-ene VPA (containing 5% (Z)-2-ene VPA), and ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) and an equivalent amount of IN NaOH were combined and made to 10 mL with d i s t i l l e d water to y i e l d the main stock s o l u t i o n . A 200 uL a l i q u o t of the main stock s o l u t i o n was added to 5 mL of control t i s s u e homogenate to y i e l d stock t i s s u e s o l u t i o n #1. D i l u t i o n of stock t i s s u e s o l u t i o n #1, as o u t l i n e d i n t a b l e 4 , with control t i s s u e homogenate a f f o r d s the samples f o r the high concentration range c a l i b r a t i o n curve. The concentration ranges f o r each compound quantitated are l i s t e d i n t a b l e 5. High concentration range c a l i b r a t i o n curves were prepared f o r each t i s s u e assayed by s p i k i n g a 200 uL a l i q u o t of main stock s o l u t i o n i n t o the various t i s s u e s analyzed followed by the d i l u t i o n scheme i n t a b l e 4. Stock t i s s u e s o l u t i o n #2 was made by s p i k i n g 50 uL of stock t i s s u e s o l u t i o n #1, varying amounts of each m e t a b o l i t e , and an equivalent amount of IN NaOH corresponding to the amount of metabolites added i n t o approximately 5 mL of control t i s s u e . D i l u t i o n of stock t i s s u e s o l u t i o n #2, as d e t a i l e d i n t a b l e 4, y i e l d s the samples f o r the low concentration range c a l i b r a t i o n curves f o r VPA and i t s m e t a b o l i t e s . The concentration ranges f o r each compound are l i s t e d i n t a b l e 5. S i m i l a r i l y , the low concentration range c a l i b r a t i o n curves were prepared f o r each t i s s u e assayed by using control t i s s u e to d i l u t e a l i q u o t s of stock t i s s u e s o l u t i o n #1. The c a l i b r a t i o n curves used f o r assaying the amount of free (E ,E)-and ( E , Z ) - 2 , 3 ' - d i e n e VPA present i n the plasma u l t r a f i l t r a t e were prepared by using d i s t i l l e d water f o r d i l u t i o n of the stock s o l u t i o n s . The f i n a l concentrations obtained were i d e n t i c a l to those l i s t e d i n t a b l e 5. Table 4: Volumes of control t i s s u e homogenate used f o r the d i l u t i o n of stock t i s s u e homogenate containing VPA and VPA metabolites to provide the samples required to produce a c a l i b r a t i o n curve. Sample uL of Stock Solution uL of Control Tissue Total Volume uL 1 0 250 250 2 50 200 250 3 100 150 250 4 150 100 250 5 200 50 250 6 250 0 250 tn O Table 5: Concentrations f o r the low and high range c a l i b r a t i o n curves f o r VPA and i t s m e t a b o l i t e s . COMPOUND LOW RANGE HIGH RANGE VPA 0 - 2.00 ug/mL 0 - 200.00 ug/mL ( E , E ) - 2 , 3 ' - d i e n e VPA 0 - 1.99 ug/mL 0 - 199.40 ug/mL ( E , Z ) - 2 , 3 ' - d i e n e VPA 0 - 160 ng/mL 0 - 15.95 ug/mL (E)-2-ene VPA 0 - 2.01 ug/mL 0 - 200.80 ug/mL (Z)-2-ene VPA 0 - 261 ng/mL 0 - 26.10 ug/mL 3-ene VPA 0 - 3.94 ug/mL 4-ene VPA 0 - 301 ng/mL ( E ) - 2 , 4 - d i e n e VPA 0 - 4.60 ug/mL 3-keto VPA 0 - 4.10 ug/mL 4-keto VPA 0 - 4.08 ug/mL 3-OH VPA 0 - 2.83 ug/mL 4-OH VPA 0 - 11.46 ug/mL 5-OH VPA 0 - 6.54 ug/mL 2-PGA 0 - 3.98 ug/mL 2.5 .4 E x t r a c t i o n and D e r i v a t i z a t i o n To a 250 uL a l i q u o t of t i s s u e homogenate or plasma or to 250 mg a l i q u o t of each pooled brain s e c t i o n homogenate, 50 uL of i n t e r n a l standard s o l u t i o n , 100 uL of d i s t i l l e d water, and 55 uL of IN HCl were added. The samples were v i g o r o u s l y mixed and extracted twice by gentle mechanical r o t a t i o n with 1 mL of ethyl acetate f o r 20 minutes. A f t e r each e x t r a c t i o n step the samples were c e n t r i f u g e d f o r 10 min at 1000 g . To the combined organic f r a c t i o n s , 400 uL of IN NaOH was added and extracted by gentle mechanical r o t a t i o n f o r 10 minutes. The organic l a y e r was discarded and 85 uL of 4N HCl was added to the aqueous p o r t i o n to y i e l d a pH of 1 - 2. The a c i d i c f r a c t i o n was then extracted with 1 mL of ethyl acetate by gentle mechanical r o t a t i o n f o r 10 min. Upon s e p a r a t i o n , the organic l a y e r was d r i e d over anhydrous Na2S04 and the volume reduced to 50 uL with n i t r o g e n . The e x t r a c t was then t r a n s f e r r e d i n t o a 1 mL R e a c t i - V i a l ^ . To the r e s i d u e , 10 uL of d i i s o p r o p y l e t h y l a m i n e , 10 uL of quinol (1 mg/mL i n ethyl a c e t a t e ) , and 10 uL of a - b r o m o - 2 , 3 , 4 , 5 , 6 -pentafluorotoluene (PFB, 30% i n ethyl acetate) were added and the mixture heated f o r 45 minutes at 60°C. The sample was cooled and 50 uL of N - m e t h y l - 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 a m i d e (MSTFA) was added and heated f o r another 45 minutes at 60°C. Once c o o l , the d e r i v a t i z e d e x t r a c t s were transfered i n t o 100 uL l i m i t e d volume i n s e r t s which were placed i n t o an auto-sample v i a l and sealed with an aluminum crimp cap. A 1 uL a l i q u o t was i n j e c t e d v i a an autosampler. 2 . 5 . 5 E x t r a c t i o n E f f i c i e n c y The amount of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA extracted from brain t i s s u e homogenate was evaluated. S o l u t i o n s i n ethyl acetate were prepared by the combined a d d i t i o n of varying amounts of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n t o dry ethyl acetate such that the f i n a l concentration of each compound was 0 . 4 , 0 . 8 , 1 . 2 , 1 . 6 , and 2.0 ug/mL. S i m i l a r s o l u t i o n s at various concentrations of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n control b r a i n t i s s u e homogenate were prepared. A 250 uL a l i q u o t of b r a i n t i s s u e homogenate spiked with VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA at each concentration was e x t r a c t e d , according to the aforementioned procedure. To the extracted samples, i n t e r n a l standards i n ethyl acetate were added and the mixtures d e r i v a t i z e d and assayed based on the p r e v i o u s l y d e t a i l e d procedure. A 250 uL a l i q u o t from each sample concentration c o n t a i n i n g VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n ethyl acetate was s i m i l a r i l y d e r i v a t i z e d and assayed. The peak area r a t i o s obtained f o r the standard samples of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA d i s s o l v e d i n ethyl acetate were used to generate a standard curve. The concentrations of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA extracted from brain t i s s u e homogenate were then determined from t h i s standard curve. The r a t i o between the observed concentration i n b r a i n t i s s u e homogenate to the t h e o r e t i c a l concentration f o r each compound was used to determine the percent of compound extracted out of the brain t i s s u e , i.e. the e x t r a c t i o n e f f i c i e n c y . 2.6 CALCULATIONS AND STATISTICS 2 . 6 . 1 C a l c u l a t i o n of Pharmacokinetic Parameters The pharmacokinetic parameters such as systemic clearance ( C l ) , h a l f - l i f e ( t 1 / 2 ) , apparent e l i m i n a t i o n r a t e constant ( K e ) , and apparent volume of d i s t r i b u t i o n (Vj) were c a l c u l a t e d using the equations of G i b a l d i and P e r r i e r (1982). The area under the plasma and t i s s u e concentration-time curves were determined by the t r a p e z o i d a l r u l e ( G i b a l d i and P e r r i e r , 1982). Because the apparent e l i m i n a t i o n rate constant was not a v a i l a b l e , the area under the curve a f t e r the l a s t sample measured could not be c a l c u l a t e d . Therefore, the area was expressed as area under the curve from 0 to 10 hours ( A U C o - i o h ) - The plasma clearances f o r each drug were then reported as CT0-lOh• Since the b i o a v a i l a b i l i t y (F) f o r each compound was not determined, clearance was expressed as C l o - i o / F . The p r e c i s i o n of the data was expressed as ± S.D. 2 . 6 . 2 S t a t i s t i c a l A n a l y s i s D i f f e r e n c e s i n the normally d i s t r i b u t e d v a r i a b l e s , i.e. pharmacokinetic parameters, were assessed by one-way a n a l y s i s of v a r i a n c e . I f a s i g n i f i c a n t F r a t i o was encountered, an a d d i t i o n a l t e s t , Newman K e u l ' s mutiple comparison t e s t , was performed to determine which group was s i g n i f i c a n t l y d i f f e r e n t . A l l s t a t i s t i c a l t e s t s used a P-value of 0.05 to assess s i g n i f i c a n c e . The ED50 values from the dose-response curves were determined using the method of L i t c h f i e l d and W i l c o x i n (1948). Differences between the ED50 values f o r each compound administered were assessed using the method of C h i - s q u a r e s . 3 . RESULTS 3.1 CHEMICAL SYNTHESES 3.1.1 2 - n - P r o p y l - ( E)-2 - P e n t e n o i c Acid ((E)-2-ene VPA) Following the procedure o u t l i n e d i n scheme 1, 2 - n - p r o p y l - ( E)-2-pentenoic a c i d was synthesized i n good y i e l d . The f i n a l product was found to contain two stereoisomers, both i d e n t i f i e d by GC-MS as methyl e s t e r d e r i v a t i v e s of 2-n-propyl-(Z)-2-pentenoic a c i d and 2 - n - p r o p y l - ( E ) -2-pentenoic a c i d ( f i g u r e 5 ) . The c o n f i g u r a t i o n of the two isomers was confirmed by NMR ( f i g u r e 6 ) . The mass spectrum of methyl 2 - n - p r o p y l - ( E ) -2-pentenoate revealed a prominent ion at m/z 156 corresponding to the molecular i o n . In a d d i t i o n , a fragment ion at m/z 141 represented the l o s s of the methyl e s t e r moiety, [M-15] + . From the NMR spectrum, the m a j o r i t y of the product possessed the ( E ) - c o n f i g u r a t i o n . A comparison of the i n t e g r a t e d peaks at 6.8 ppm and at 5.9 ppm, corresponding to the proton at C3 f o r the (E)- and (Z)-isomers r e s p e c t i v e l y , i n d i c a t e d that the product was 95% 2- n - p r o p y l - ( E)-2- p e n t e n o i c a c i d . 3.1.2 2 - ( ( Z ) - l ' - P r o p e n y l ) - ( E)-2 - P e n t e n o i c A c i d ( ( E , Z)-2 , 3 ' diene VPA) The method used f o r the synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) -2 -pentenoic a c i d (scheme 2) was s i m i l a r to that of 2 - n - p r o p y l - ( E)-2-pentenoic a c i d . The y i e l d obtained from the synthesis of 2 - ( ( Z ) - l ' -propenyl)-(E)-2-pentenoic a c i d s t a r t i n g with the /3-hydroxy-0',7'-unsaturated e s t e r was 85%. The product was i d e n t i f i e d by GC-MS as the methyl e s t e r d e r i v a t i v e ( f i g u r e 7) . Major ions at m/z 154 and 139 COOEt COOEt COOH ISOMER E-ISOMER Synthesis of (E)-2-ene VPA: a=EtOH, H2SO4, benzene, A; b=[(CH3)2CH]2NH, n - B u L i , CH3CH 2CH0, THF, -78 e C; c=(C 2 H 5 ) 3 N, C H 3 S0 2 C1, C H 2 C1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, H C 1 . 57 Figure 5: Total ion chromatogram and mass spectra of methyl 2-n-propyl-(E)-2-pentenoate and methyl 2-n-propyl-(Z)-2-pentenoate . C H 2 - C H HO^  sS> CH 3 -CH 2 -QH CH 3 -CH 2 -CH V I I I ' I 1 I 1 I ' 1 ' 1 ' I ' I ' ! 1 I 1 I ' I 1 1 ' 1 '—"1 " 1 ' 1 1 1 1 1 1 [ 1 ; 1 1 1 1 1 1 1 1 1 1 1 j 1 1 , 1 , ~ 7 - & 6 . 8 6 . 6 S.i 6 . 2 6 . a S . B 5 . 6 5 . 4 5 . 2 5 . 8 4. 8 4. 6 4. 4 4. 2 4 . 8 3 . 8 3 . 6 3 . 4 3 . 2 3 . 8 2 . 8 2 . 6 2 . 1 2 . 2 2 . 8 1 8 1 6 1 4 1 2 I I B P P M Figure 6: 400 MHz Proton NMR spectrum of 2-n-propyl-(E)-2-pentenoic acid (*=2-n-propyl-(Z)-2-pentenoic a c i d ) . C O COOEt COOEt Scheme 2: Synthesis of ( E , Z ) - 2 , 3 ' - d i e n e VPA: a=EtOH, H2SO4, benzene, A; b=[(CH3)2CH]2NH, n - B u L i , CH3CH2CH0, THF, -78'C; c=(C 2 H5) 3 N, CH 3 S0 2 C1, C H 2 C 1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, H C l . 36 93 Scan f*33 Figure 7: Total ion chromatogram and mass spectrum of methyl 2-((Z)-1'-propenyl)-(E)-2-pentenoate. corresponded to the molecular ion and l o s s of the methyl e s t e r d e r i v a t i v e , r e s p e c t i v e l y . The chemical s t r u c t u r e was confirmed by NMR ( f i g u r e 8) to be 95% i s o m e r i c a l l y pure f o r the 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) -2-pentenoic a c i d . 3 . 1 . 3 2 ~ ( ( E ) - l ' - P r o p e n y l ) - ( E ) - 2 - P e n t e n o i c Acid ( ( E , E ) - 2 , 3 ' - d i e n e VPA) The synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was based on the procedures of Lee et al. (1989)(scheme 3 ) . The n u c l e o p h i l e , potassium h y d r i d e , was used to e l i m i n a t e the mesyl group to y i e l d the a , ^ - u n s a t u r a t e d e s t e r . The synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d had been completed before the a v a i l a b i l i t y of DBU as was used i n the synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . To obtain s u f f i c i e n t material f o r the animal experiments, the synthesis of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was repeated several times to a f f o r d several grams of product. The f i n a l product was i d e n t i f i e d as the methyl e s t e r d e r i v a t i v e f o l l o w i n g GC-MS a n a l y s i s ( f i g u r e 9 ) . From the mass spectrum, prominent ions at m/z 154, 139, and 95 corresponded to a molecular i o n , l o s s of a methyl group, and [M-C00CH3]+, r e s p e c t i v e l y . The NMR spectrum f o r 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d ( f i g u r e 10) compared favorably to that of published values (Acheampong and Abbott, 1985; Lee et a 7 . , 1989). The product was found to be 95 - 97% i s o m e r i c a l l y pure based on GC-MS and NMR d a t a . CH, C H 2 - C H i 1 i 1 i ' i ' i • i 1 i—'—i—1—i—1—i—•—i—1—i—>—i—<—\—'—i—i—i—i—i—i—i—<—i—i—i—'—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—,—,—,—i—,—,— 7 ( i 6 .B 6 .6 6.4 6 .2 6.0 5.8 5 .6 5.4 5.2 5.8 4 .B 4 .6 4.4 fl. 2 4. 0 3. 8 3. 6 3.4 3 .2 3 .8 2 .8 2. 6 2.4 2.2 2. 8 1 8 1 6 1 i 1 2 1 0 PPM Figure 8: 400 MHz Proton NMR spectrum of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d (*=2-((E)-T propenyl)-(E)-2-pentenoic a c i d ) . as PO 63 E.Z-ISOMER E.E-ISOMER Scheme 3 : Synthesis of ( E , E ) - 2 , 3 ' - d i e n e VPA: a=K2C03, E t I , 18-crown-6, THF, A; b=[(CH3)2CH]2NH, n - B u L i , HMPA, CH3CH2CHO, THF, - 7 8 ° C ; c=(C 2 H 5 ) 3 N, CH3S02C1, CH 2 C1 2 , 0 ° C ; d=DBU, THF, A; e=NaOH, A, HC1. 64 Figure 9: Total ion chromatogram and mass spectrum of methyl 2-((E)-1'-propenyl)-(E)-2-pentenoate. C H , - C H C H = C H | C H = C H C H 3 - C H C H J C H 3 — C H 2 I, I 1 1 1 1 1 1 1 1 1 j 1 1 1 4 3 1 1 1 1 1 1 ' 1 1 I 1 1 ' 1 I 1 1 1 1 1 1 1 1 1 j 1 1 2 1 0 P P M Figure 10: 300 MHz Proton NMR spectrum of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d ( * = 2 - ( ( Z ) - l ' propenyl)-(E)-2-pentenoic a c i d ) . v u ' 3.2 ASSAY DEVELOPMENT 3 . 2 . 1 E x t r a c t i o n and D e r i v a t i z a t i o n The l i v e r , h e a r t , kidney, l u n g , and brain samples were uniformly homogenized i n d i s t i l l e d water before e x t r a c t i o n . The absolute e x t r a c t i o n e f f i c i e n c i e s f o r VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n brain t i s s u e homogenates were w i t h i n acceptable l i m i t s (table 6 ) . The l i m i t s of d e t e c t i o n achieved f o r VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' -diene VPA i n b r a i n t i s s u e homogenate (13 - 38 ng/mL) were 10 - 30 pg with a s i g n a l - t o - n o i s e r a t i o of l e s s than 3. S i m i l a r l i m i t s of d e t e c t i o n were observed f o r these compounds i n the other t i s s u e s examined. A two step d e r i v a t i z a t i o n procedure was employed to ensure good chromatographic separation of a l l compounds of i n t e r e s t from that of the background. A pentafluorobenzyl (PFB) e s t e r on the c a r b o x y l i c a c i d group was i n i t i a l l y formed and was immediately followed by a second d e r i v a t i v e to y i e l d the t r i m e t h y l s i l y l (TMS) e s t e r f o r those compounds possessing a hydroxyl or oxo moiety. The d e r i v a t i z a t i o n of the c a r b o x y l i c a c i d group by the PFB reagent proceeded to completion since there was no t r a c e of a TMS e s t e r present f o r each compound assayed. That i s , i f the i n i t i a l d e r i v a t i z a t i o n of the c a r b o x y l i c a c i d group was not complete, the a d d i t i o n of the second d e r i v a t i z i n g reagent (MSTFA) would have reacted with the remaining f r e e a c i d to y i e l d the TMS e s t e r . S i m i l a r l y , d e r i v a t i z a t i o n of the hydroxyl or 3-keto groups with TMS proceeded to completion since no ions were detected f o r these p o l a r compounds at [M-181]", / . e . l o s s of the PFB group from metabolites where the hydroxyl or 3-keto moiety was u n d e r i v a t i z e d . Table 6: The e x t r a c t i o n e f f i c i e n c i e s of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' -diene VPA from brain t i s s u e homogenate at various concentrations (n=3). Concentration (ug/mL) VPA 0.4 0 . 8 1.2 1.6 2.0 (E)-2-ene VPA 0.4 0 . 8 1.2 1.6 2.0 ( E , E ) - 2 , 3 ' - d i e n e VPA 0.4 0 . 8 1.2 1.6 2.0 E x t r a c t i o n E f f i c i e n c y (%,S.D.) 85±6 73±3 80±4 77±1 82±1 82±23 84±4 77±2 80±3 76±5 87±4 82±25 59±5 60±4 67±6 55±6 61±8 57±21 3 . 2 . 2 C a l i b r a t i o n Curves The c a l i b r a t i o n curves obtained f o r VPA and the metabolites of i n t e r e s t from b r a i n t i s s u e homogenates showed l i n e a r i t y w i t h i n the concentration ranges used ( f i g u r e s 11 - 20). Although not shown, s i m i l a r r e s u l t s f o r c a l i b r a t i o n curves were obtained from the other t i s s u e homogenates and plasma. The i n t e r - a s s a y v a r i a t i o n was assessed based on the slopes of each c a l i b r a t i o n curve i n each t i s s u e assayed over an 18 month period and were found to be acceptable (table 7) . The compounds demonstrating the best o v e r a l l assay p r e c i s i o n were those having a deuterated analogue of t h e i r respective s t r u c t u r e as the i n t e r n a l standard, i . e . VPA, (E)-2-ene VPA, and 3-keto VPA. Compounds quantitated without a s p e c i f i c deuterated analogue as the i n t e r n a l standard tended to have a greater r e l a t i v e standard d e v i a t i o n . Since a c a l i b r a t i o n curve f o r each compound of i n t e r e s t was generated i n - c o n j u n c t i o n with each set of unknown samples, good i n t e r - a s s a y v a r i a b i l i t y was not a c r i t i c a l f a c t o r . 3 . 2 . 3 Detection of VPA and I t s Metabolites The d e t e c t i o n of VPA and i t s metabolites was by negative ion chemical i o n i z a t i o n gas chromatography-mass spectrometry (NICI GC-MS) using s e l e c t e d - i o n - m o n i t o r i n g (SIM). This method of i o n i z a t i o n i s a gentle technique and as a r e s u l t fragmentation of d e r i v a t i z e d compounds was minimal. The fragment ion monitored f o r each compound was the [M-181]" base ion from l o s s of the PFB group from the d e r i v a t i z e d c a r b o x y l i c a c i d (table 8 ) . The mass chromatograms generated from the SIM of each compound of i n t e r e s t from brain t i s s u e homogenate are i l l u s t r a t e d i n f i g u r e 21. 69 O or or < < 2.00--0.00 25.00 50.00 75.00 100.00 125.00 150.00 175.00 200.00 VPA CONCENTRATION (ug/mL) o or: < UJ or < 0.000 0.00 0.20 0.40 0.60 0.80 1.00 VPA CONCENTRATION (ug/mL) Figure 11: High and low range concentration cal ibrat ion curves for VPA in brain tissue homogenate. 70 16.00-r 0.00 25.00 50.00 75.00 100.00 125.00 150.00 175.00 200.00 ( E , E ) - 2 , 3 ' - d i e n e VPA CONCENTRATION ( u g / m L ) 6.00-r 0.00 0.20 0.40 0.60 0.80 1.00 ( E , E ) - 2 , 3 ' - d i e n e VPA CONCENTRATION ( u g / m L ) Figure 12: High and low range concentration c a l i b r a t i o n curves f o r ( E , E ) - 2 , 3 ' - d i e n e VPA i n rat brain t i s s u e homogenate. 71 7.00 -r 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 ( E , Z ) - 2 , 3 , - d i e n e VPA CONCENTRATION ( u g / m L ) 1.25-r 0.00 0.02 0.04 0.06 0.08 ( E , Z ) - 2 , 3 , - d i e n e VPA CONCENTRATION ( u g / m L ) Figure 13: High and low range concentration c a l i b r a t i o n curves f o r ( E , Z ) - 2 , 3 ' - d i e n e VPA i n r a t brain t i s s u e homogenate. 72 Figure 14: C a l i b r a t i o n curve f o r ( E ) - 2 , 4 - d i e n e VPA i n r a t brain t i s s u e homogenate. 73 0.00 25.00 50.00 75.00 100.00 125.00 150.00 175.00 200.00 225.00 ( E ) - 2 - e n e VPA CONCENTRATION ( u g / m L ) 5.00-r 0.00 0.20 0.40 0.60 0.80 1.00 ( E ) - 2 - e n e VPA CONCENTRATION ( u g / m L ) Figure 15: High and low range concentration c a l i b r a t i o n curves f o r (E)-2-ene VPA i n brain t i s s u e homogenate. 74 O < < LU or: < < Ld CL 2.50 T 2.00 1.50 + 1.00 + 0.50 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 ( Z ) - 2 - e n e VPA CONCENTRATION ( u g / m L ) o !< or < or < 0.300 -r 0.250 0.200 + 0.150 0.100 + 0.050 + 0.000 0.000 0.025 0.050 0.075 0.100 0.125 0.150 ( Z ) - 2 - e n e VPA CONCENTRATION ( u g / m L ) Figure 16: High and low range concentration c a l i b r a t i o n curves f o r (Z)-2-ene VPA i n r a t brain t i s s u e homogenate. 75 O cr < Ld cr < < Ld CL 0.00 0.00 1.00 2.00 3.00 4.00 3 - e n e VPA CONCENTRATION ( u g / m L ) < cr < Ld cr < Q_ o.ooo 0.00 0.03 0.05 0.08 0.10 0.12 0.15 0.18 0.20 0.22 4 - e n e VPA CONCENTRATION ( u g / m L ) Figure 17: C a l i b r a t i o n curves f o r 3-ene VPA and 4-ene VPA i n r a t b r a i n t i s s u e homogenate. 76 0.00 1.00 2.00 3.00 4.00 5.00 3-ke to VPA CONCENTRATION (ug/mL) 0.00 0.25 0.50 0.75 1.00 1.25 4-ke to VPA CONCENTRATION (ug/mL) Figure 18: C a l i b r a t i o n curves f o r 3-keto VPA and 4-keto VPA i n r a t b r a i n t i s s u e homogenate. 77 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3 - O H VPA CONCENTRATION ( u g / m L ) 0.00 0.10 0.20 0.30 0.40 0.50 4 - O H VPA CONCENTRATION ( u g / m L ) Figure 19: C a l i b r a t i o n curves f o r 3-OH VPA and 4-OH VPA i n r a t b r a i n t i s s u e homogenate. 78 3.00-r 0.00 0.25 0.50 0.75 1.00 1.25 1.50 5 - O H VPA CONCENTRATION ( u g / m L ) 1.00 T 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 2 - P G A CONCENTRATION ( u g / m L ) Figure 20: C a l i b r a t i o n curves f o r 5-OH VPA and 2-PGA i n r a t brain t i s s u e homogenate. Table 7: Inter-assay v a r i a t i o n based on the slopes of the c a l i b r a t i o n curves over an 18 month period f o r VPA and i t s m e t a b o l i t e s . ()-n BRAIN PLASMA LIVER KIDNEY HEART (10) (9) (in (7) (8) ( E , E ) - 2 , 3 ' - d i e n e VPA 13.9% 15.0% 7.7% 1.7% 12.3% ( E , Z ) - 2 , 3 ' - d i e n e VPA 13.3 8 . 0 5.3 1.1 6 . 0 ( E ) - 2 , 4 - d i e n e VPA 17.9 12.7 11.1 18.4 6.1 (E)-2-ene VPA 9.1 2.9 3 . 8 8.7 2.1 (Z)-2-ene VPA 13.4 8.2 16.2 9.3 12.6 3-ene VPA 16.6 9.4 20.1 7 .5 2.1 4-ene VPA 17.2 4 . 8 19.1 7.2 7.7 VPA 5.5 10.8 0.9 2.7 7.9 4-keto VPA 18.7 16.9 23.0 1.5 5.0 3-keto VPA 6.2 2.4 13.1 2.2 8 . 9 3-OH VPA 13.3 20.8 21.6 9.6 14.0 4-OH VPA 1.3 5.6 3.6 11.6 1.7 5-OH VPA 10.1 13.0 15.5 15.9 3 . 0 2-PGA 15.1 10.2 11.4 3 . 6 9.3 Table 8: Ions monitored f o r the q u a n t i t a t i v e a n a l y s i s and the r e t e n t i o n times of i n t e r n a l s t a n d a r d s ( * ) , VPA and i t s m e t a b o l i t e s . COMPOUNDS ION MONITORED (m/z) RETENTION TIME (MINUTES) 4-ene VPA 141 7.48 [2H6]VPA* 149 7.50 VPA 143 7.55 3-ene VPA 141 7.66 (Z)-2-ene VPA 141 7.78 [ 2 H 3 ]2-ene VPA* 144 8.62 (E)-2-ene VPA 141 8.65 ( E , Z ) - 2 , 3 ' - d i e n e VPA 139 8.47 ( E ) - 2 , 4 - d i e n e VPA 139 8.78 ( E , E ) - 2 , 3 ' - d i e n e VPA 139 9.52 4-keto VPA 157 10.27 3-OH VPA 231 11.49 4-OH VPA 231 11.20/11.77 [ 2 H 3 ]3-keto VPA* 232 12.77 3-keto VPA 229 12.80 5-OH VPA 231 13.31 2-MGA* 325 19.15 2-PGA 353 21.23 1 353. J 143 325. 353. 325 - I , 1 1 r———I : 1 1 1 8 10 12 14 16 18 20 22 10 12 14 16 — I ' 1— 18 20 22 TIME (MINUTES) Figure 21: Mass chromatograms of VPA, VPA metabolites, and i n t e r n a l standards i n r a t brain homogenate f o l l o w i n g i . p . administration of 150 mg/kg of VPA (1) , and mass chromatograms of i n t e r n a l standards added to control r a t brain homogenate (2): a=4-ene VPA (7.48 m i n . ) ; b=3-ene VPA (7 .66); c=(Z)-2-ene VPA (7 .78); d=(E)-2-ene VPA ( 8 . 6 5 ) ; e=D6-VPA (7 .49); f=VPA ( 7 . 5 5 ) ; g=(E,Z)-2,3'-diene VPA ( 8 . 4 7 ) ; h=(E)-2,4-diene VPA (8.78); i = ( E , E ) - 2 , 3 ' - d i e n e VPA ( 9 . 5 2 ) ; j=D3-(E)-2-ene VPA ( 8 . 6 2 ) ; k=4-keto VPA (10.27); l/n=4-0H VPA (11.20/11.77); m=3-0H VPA (11.49); o=5-0H VPA (13.31); p=D3-3-keto VPA (12.77); q=3-keto VPA (12.80); r=2-M6A (19.15); s=2-PGA (21.23). 00 These s e l e c t e d ion mass chromatograms were well resolved and f r e e of any i n t e r f e r i n g endogenous substances. Comparable r e s u l t s were obtained f o r the plasma and other t i s s u e e x t r a c t s . 3 . 3 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF VPA 3 . 3 . 1 P r o f i l e of VPA i n Plasma Following i . p . a d m i n i s t r a t i o n of 150 mg/kg of VPA to r a t s , the drug was r e a d i l y absorbed from the p e r i t o n e a l c a v i t y , reaching peak plasma ( C m a x ) concentrations w i t h i n 30 minutes. The apparent maximum plasma concentration was 142.7 ± 51.8 ug/mL. The e l i m i n a t i o n of VPA from plasma d i d not f o l l o w a t y p i c a l l o g - l i n e a r d e c l i n e but was i n t e r r u p t e d by a temporary r i s e i n plasma l e v e l s that occurred 4 - 6 hours f o l l o w i n g drug a d m i n i s t r a t i o n ( f i g u r e 22). The area under the curve (AUC) f o r VPA i n plasma measured from 0 -10 hours was c a l c u l a t e d to be 455 ± 68 ug*h/mL. Plasma clearance of VPA was c a l c u l a t e d to be 92 ± 14 mL/h and was expressed as Cln-ioh/F since the b i o a v a i l a b i l i t y (F) f o r VPA a f t e r i . p . a d m i n i s t r a t i o n i n the r a t was not known. 3 . 3 . 2 P r o f i l e of VPA i n Peripheral Tissues The k i n e t i c p r o f i l e of VPA was determined i n l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g i . p . a d m i n i s t r a t i o n . The d i s t r i b u t i o n of VPA from the plasma to the t i s s u e s was not r a p i d with maximal t i s s u e concentrations o c c u r r i n g w i t h i n 30 - 45 minutes a f t e r i n j e c t i o n . The cmax values f o r VPA i n the l i v e r , kidneys, h e a r t , and lungs were 83 1.0 -\ 1 1 1 0 200 400 600 TIME (MINUTES) Figure 22: VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i - P -a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . 225.6 ± 63.1, 179.6 ± 32.0, 52.7 ± 13.3, and 93.2 ± 13.0 ug/g r e s p e c t i v e l y . The e l i m i n a t i o n p r o f i l e s of VPA i n these t i s s u e s were s i m i l a r to that seen i n plasma i n c l u d i n g the temporary increase i n VPA l e v e l s that occurred 4 - 6 hours f o l l o w i n g drug a d m i n i s t r a t i o n ( f i g u r e 22). The concentration of VPA was highest i n the l i v e r and remained that way f o r the e n t i r e 10 hour p e r i o d . The AUCo-ioh values f o r the l i v e r , k i d n e y s , h e a r t , and lungs were c a l c u l a t e d to be 854 ± 124, 698 ± 114, 138 + 25, and 229 ± 39 ug h/g r e s p e c t i v e l y . A comparison of the tissue/plasma r a t i o s at t m a x showed that VPA appeared to concentrate mainly i n the l i v e r and kidneys with r a t i o s to plasma values of 1.8 and 1.3 r e s p e c t i v e l y . At tjoh» the l i v e r / p l a s m a r a t i o increased to 4.6 while i n kidneys the r a t i o increased s l i g h t l y to 1.8. Thus, VPA appears to p e r s i s t i n the l i v e r and kidney t i s s u e s independent of plasma l e v e l s . The tissue/plasma r a t i o of VPA i n the heart and lungs at t m a x was c a l c u l a t e d to be 0.3 and 0.9 r e s p e c t i v e l y . These r a t i o s decreased to 0.02 and 0.2, r e s p e c t i v e l y at 10 hours. 3.3.3 P r o f i l e of VPA i n Brain The k i n e t i c p r o f i l e of VPA i n b r a i n t i s s u e was determined i n the whole b r a i n (WB) and the f o l l o w i n g brain r e g i o n s : hippocampus (HIP), s u p e r i o r c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), and the caudate putamen (CP) ( f i g u r e 23). The i s o l a t i o n of c e r t a i n areas of the brain may i n d i c a t e regional d i f f e r e n c e s i n the k i n e t i c behavior of a compound that may otherwise be overlooked upon whole b r a i n a n a l y s i s . 85 CD O cn Z5 < Q_ > 100.00 — 10.00 - -1.00 - -0.10 0 100 200 300 400 500 600 TIME (MINUTES) Figure 23: VPA concentration-time curves i n r a t plasma (PLA), whole b r a i n (WB), hippocampus (HIP), superior c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), and putamen (CP) f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (WB,n=8/time p o i n t ; brain regions were pooled,n=8/time p o i n t ) . The d i s t r i b u t i o n of VPA i n t o the brain was r e l a t i v e l y f a s t with peak l e v e l s i n a l l regions a t t a i n e d w i t h i n 15 - 30 minutes. The concentration of VPA i n the whole brain at t m a x was 5 0 . 6 ± 1 3 . 0 ug/g w h i l e i n the various brain regions the concentrations were between 4 5 . 1 - 6 9 . 3 ug/g. The d i s t r i b u t i o n of VPA throughout the b r a i n appeared to be homogeneous. The e l i m i n a t i o n p r o f i l e of VPA from the b r a i n was s i m i l a r to that i n plasma with the r e c y c l i n g most evident f o r the b r a i n f r a c t i o n s . The b r i e f increase i n VPA concentration observed i n plasma a l s o occurred i n the b r a i n but to a l e s s e r degree. The A U C o - i o h f o r VPA i n whole b r a i n was 105 ± 23 ug*h/mL whereas the A U C o - i o h i n the various regions of the b r a i n were between 104.6 - 132.6 ug*h/mL. Of a l l the t i s s u e s assayed, i n c l u d i n g peripheral t i s s u e s , the brain/plasma r a t i o f o r VPA was the lowest. The r a t i o of whole brain/plasma at t m a x was 0 . 3 while the tissue/plasma r a t i o s f o r the i n d i v i d u a l b r a i n s e c t i o n s were 0 . 3 - 0 . 4 . Ten hours f o l l o w i n g the a d m i n i s t r a t i o n of VPA, these r a t i o s were c a l c u l a t e d to be i n the range of 0 . 0 1 - 0 . 0 3 . The brain/plasma r a t i o s between whole brain sample and the i n d i v i d u a l b r a i n s e c t i o n s appeared to be s i m i l a r . 3.4 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E)-2-ENE VPA 3.4.1 P r o f i l e of (E)-2-ene VPA i n Plasma Upon s i n g l e dose i . p . a d m i n i s t r a t i o n of (E)-2-ene VPA, peak plasma concentrations of 143.0 ± 5 0 . 0 ug/mL were a t t a i n e d w i t h i n 30 minutes. The e l i m i n a t i o n p r o f i l e of (E)-2-ene VPA from plasma was i n t e r r u p t e d by an increase i n the plasma l e v e l 4 - 6 hours a f t e r drug a d m i n i s t r a t i o n ( f i g u r e 24). T h e r e a f t e r , the plasma concentration of (E)-2-ene VPA maintained a plateau or e q u i l i b r i u m phase that appeared to continue w e l l past the l a s t sample at 10 hours. The AUCo-ioh ° f (E)-2-ene VPA i n plasma was determined to be 497 + 38 ug'h/mL. Plasma clearance f o r (E)-2-ene VPA expressed as ClQ-iOh/F w a s 82 ± 6 mL/h. 3 . 4 . 2 P r o f i l e of (E)-2-ene VPA i n Peripheral Tissues The absorption and e l i m i n a t i o n p r o f i l e s of (E)-2-ene VPA i n the l i v e r , kidneys, h e a r t , and lungs were s i m i l a r to that i n plasma ( f i g u r e 24). The time taken to reach peak t i s s u e l e v e l s was 30 - 45 minutes f o l l o w i n g a d m i n i s t r a t i o n . The C m a x values i n the l i v e r , kidneys, h e a r t , and lungs were 244.5 ± 6 4 . 9 , 99.5 ± 2 9 . 5 , 66.3 ± 2 9 . 8 , and 52.8 ± 17.8 ug/g r e s p e c t i v e l y . Although the concentration of (E)-2-ene VPA i n the l i v e r was i n i t i a l l y higher than that of plasma the AUCo-ioh ° f 3 8 4 ± 6 3 ug-h/mL f o r the l i v e r was l e s s than that of plasma. Thus w h i l e the l i v e r / p l a s m a r a t i o at t m a x was 1 .4 , at t j o h t h e r a t i o had decreased to 0 . 5 , suggesting that (E)-2-ene VPA does not bind a v i d l y to l i v e r t i s s u e . The A U C o - i o h values f o r (E)-2-ene VPA i n the kidneys, heart and lungs were 273 ± 41, 79 ± 16, and 147 ± 18 ug*h/mL r e s p e c t i v e l y . The tissue/plasma r a t i o s determined at t m a x f o r kidneys, h e a r t , and lungs were 0 . 7 , 0 . 4 , and 0.3 r e s p e c t i v e l y . These r a t i o s decreased to 0 . 5 , 0 . 0 2 , and 0.2 at tjQh• 3 . 4 . 3 P r o f i l e of (E)-2-ene VPA i n Brain The times required to reach peak brain l e v e l s of (E)-2-ene VPA were i n the range of 15 - 30 minutes f o l l o w i n g a d m i n i s t r a t i o n . The mean cmax i n whole b r a i n was 68.8 ± 19.8 ug/g while the C m a x values observed 88 Figure 24: (E)-2-ene VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . f o r the i n d i v i d u a l pooled brain sections were between 30.0 - 52.8 ug/g. The d i s t r i b u t i o n of (E)-2-ene VPA throughout the b r a i n appeared to be homogeneous as there were no areas found to contain unusually high l e v e l s of the drug. The e l i m i n a t i o n of (E)-2-ene VPA from the whole b r a i n and various brain regions appeared to be rapid and l i n e a r f o r the i n i t i a l 4 hours f o l l o w i n g drug a d m i n i s t r a t i o n . Thereafter a temporary increase i n (E)-2-ene VPA l e v e l s occurred which was then followed by the slower e l i m i n a t i o n of the drug ( f i g u r e 25). This t r a n s i e n t r i s e i n (E)-2-ene VPA l e v e l s i n the brain corresponded to a s i m i l a r e f f e c t observed i n plasma. The AUCo-ioh value c a l c u l a t e d f o r the whole b r a i n f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n was 48 ± 10 ug*h/g. The i n d i v i d u a l pooled brain regions had AUCo-ioh values between 43 - 54 ug*h/g. The brain/plasma r a t i o f o r (E)-2-ene VPA i n the whole brain at t m a x was 0.3 which decreased to 0.04 a f t e r 10 hours. The brain/plasma r a t i o s f o r the brain s e c t i o n s were i n the range of 0.3 - 0.4 at t m a x and decreased to 0.02 -0.03 at 10 hours. 3 . 5 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E,E)-2,3'-DIENE VPA 3 . 5 . 1 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Plasma The absorption of ( E , E ) - 2 , 3 ' - d i e n e VPA i n t o the plasma c i r c u l a t i o n was r e l a t i v e l y r a p i d f o l l o w i n g i . p . a d m i n i s t r a t i o n of 150 mg/kg of the drug. The time taken to reach peak plasma l e v e l s was 15 minutes with a corresponding plasma concentration of 168.2 ± 13.9 ug/mL. The plasma l e v e l s f o r ( E , E ) - 2 , 3 ' - d i e n e VPA remained elevated f o r f i r s t hour 90 O cn Z3 < Q_ > U J z LU CN L d 100.00 10.00 — 1.00 0.10 • WB O HIP A SC A IC • CER / \ • OLF v CC • SN O MED • C P PLA o 100 200 300 400 500 600 TIME (MINUTES) Figure 25: (E)-2-ene VPA concentration-time curves i n r a t plasma (PLA), whole b r a i n (WB), hippocampus (HIP), s u p e r i o r c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), and putamen (CP) f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (WB,n=8/time p o i n t ; b r a i n regions were pooled,n=8/time p o i n t ) . f o l l o w i n g a d m i n i s t r a t i o n but t h e r e a f t e r , the diene plasma l e v e l s began to d e c l i n e ( f i g u r e 26). The e l i m i n a t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA was r a p i d although at the 4 - 6 hour period there appeared to be a b r i e f i n t e r r u p t i o n that r e s u l t e d i n a change i n the rate of d e c l i n e . The AUCrj-iOh i n plasma was 406 ± 46 ug*h/mL and the c a l c u l a t e d clearance (ClQ-10h/F) w a s 97 ± 11 mL/h. 3 . 5 . 2 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Peripheral Tissues The d i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA i n t o the l i v e r , kidneys, h e a r t , and lungs was followed a f t e r i . p . a d m i n i s t r a t i o n of the drug ( f i g u r e 26) . The times required to reach peak t i s s u e l e v e l s i n the l i v e r , kidneys, h e a r t , and lungs were i n the range of 45 - 60 minutes with corresponding t i s s u e concentrations of 76.1 ± 1 9 . 0 , 75.9 ± 6 . 7 , 42.0 ± 12.6, and 8 0 . 8 ± 15.7 ug/g r e s p e c t i v e l y . The e l i m i n a t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA from these t i s s u e s was r e l a t i v e l y r a p i d and s i m i l a r to that i n plasma. The AUCn-ioh f o r ( E , E ) - 2 , 3 ' - d i e n e VPA i n the lungs was highest at 135 ± 26 ug*h/g and lowest i n heart at 52 ± 8 ug*h/g. The A U C o - i o h values f o r l i v e r and kidneys were 133 ± 15 and 121 ± 30 ug*h/g r e s p e c t i v e l y . The tissue/plasma r a t i o s at maximum ( E , E ) - 2 , 3 ' - d i e n e VPA concentrations i n the l i v e r , kidneys, h e a r t , and lungs were 0 . 5 , 0 . 5 , 0 . 3 , and 0 . 5 , r e s p e c t i v e l y . At t ioh> the tissue/plasma r a t i o s f o r kidneys and lungs had increased to values of 1.3 and 1 . 0 , r e s p e c t i v e l y w h i l e the tissue/plasma r a t i o at 6 hours f o r both l i v e r and heart f e l l to 0 . 0 1 . At 10 hours ( E , E ) - 2 , 3 ' - d i e n e VPA i n heart and l i v e r was not r e a d i l y d e t e c t a b l e . c n I • PLASMA TIME (MINUTES) Figure 26: ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n r a t plasma, l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n (n=8/time p o i n t , e r r o r bars=S.D.) . 3.5.3 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Brain Following i . p . a d m i n i s t r a t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA, the drug q u i c k l y entered i n t o the b r a i n , reaching maximum concentrations i n most regions w i t h i n 15 minutes while i n areas l i k e the s u p e r i o r and i n f e r i o r c o l l i c u l u s and the cerebellum the t m a x was 60 minutes ( f i g u r e 27). In whole b r a i n , the t m a x was 45 minutes and corresponded to a r e l a t i v e l y high concentration of 56.2 + 8.4 ug/g. The maximum concentrations of ( E , E ) - 2 , 3 ' - d i e n e VPA i n the various pooled b r a i n s e c t i o n s f e l l between 27.6 - 57.3 ug/g. The e l i m i n a t i o n p r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA from a l l b r a i n t i s s u e s was s i m i l a r to that i n plasma, i n c l u d i n g a delay i n the d e c l i n e of ( E , E ) - 2 , 3 ' - d i e n e VPA that occurred 4 - 6 hours f o l l o w i n g drug a d m i n i s t r a t i o n . The A U C o - i o h value f o r the whole brain was 56 ± 10 ug*h/g while the i n d i v i d u a l pooled brain sections gave areas i n the range of 37 - 56 u g * h / g . The whole brain/plasma r a t i o s at t m a x and t j o h decreased from 0.3 to 0 . 1 , r e s p e c t i v e l y . However, the brain/plasma r a t i o s f o r the i n d i v i d u a l b r a i n regions at t m a x and t i o h increased with these values ranging from 0.1 - 0.3 and 0.2 - 1 .4 , r e s p e c t i v e l y . O _ J cn < C L > LxJ z U J O C N I Ld 100.00 10.00 — 1.00 — WB 13 OLF o HIP V CC A SC • SN A IC o MED • CER • C P 94 0.10 - -0.01 0 100 200 300 400 500 600 TIME (MINUTES) Figure 27: (E,E)-2,3'-diene VPA concentration-time curves in rat plasma (PLA), whole brain (WB), hippocampus (HIP), superior col l iculus (SC), infer ior co l l icu lus (IC), cerebellum (CER), olfactory bulbs (OLF), corpus callosum (CC), substantia nigra (SN), medulla (MED), and putamen (CP) following 150 mg/kg i .p . administration (WB,n=8/time point; brain regions were pooled,n=8/time point). 3 . 6 COMPARATIVE PHARMACOKINETICS AND TISSUE DISTRIBUTION OF VPA, (E)-2-ENE VPA, AND (E,E)-2,3'-DIENE VPA 3 . 6 . 1 P r o f i l e s i n Plasma The k i n e t i c p r o f i l e s of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n plasma were compared f o l l o w i n g i . p . a d m i n i s t r a t i o n of equivalent doses ( f i g u r e 28). S i m i l a r absorption 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 were noted f o r a l l three compounds during the i n i t i a l 6 hours. T h e r e a f t e r , the apparent increase i n ( E , E ) - 2 , 3 ' - d i e n e VPA e l i m i n a t i o n r e s u l t e d i n the r a p i d d e c l i n e of the diene from plasma. Common to each compound was an i n t e r r u p t i o n i n the d e c l i n e from plasma seen 4 - 6 hours f o l l o w i n g a d m i n i s t r a t i o n . This i n t e r r u p t i o n was i l l u s t r a t e d by an increase i n drug plasma concentrations that e v e n t u a l l y lead to a plateau phase. No s i g n i f i c a n t d i f f e r e n c e s were observed i n plasma C m a x values f o r the three administered compounds ( t a b l e 9) but there was a s i g n i f i c a n t d i f f e r e n c e (p<0.05) i n AUCn-ioh values between (E)-2-ene VPA and (E ,E)-2 , 3 ' - d i e n e VPA (table 10). Thus, the rate and extent of absorption of VPA d i d not d i f f e r s i g n i f i c a n t l y from that of (E)-2-ene VPA or (E ,E)-2 , 3 ' - d i e n e VPA during the 10 hour p e r i o d . 3 . 6 . 2 P r o f i l e s i n Peripheral Tissues The absorption and e l i m i n a t i o n p r o f i l e s of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA were compared i n l i v e r . From the concentration-time p l o t s , the e l i m i n a t i o n of VPA and (E)-2-ene VPA from l i v e r were comparable whereas ( E , E ) - 2 , 3 ' - d i e n e VPA appeared to c l e a r at a f a s t e r 96 1000.0 • VPA o (E ) -2 -ENEVPA • (E,E)-2 I3'-DIENE VPA 100.0 E CD Z5 o < UJ o z o o io.o 4-1.0 0.1 0 200 400 TIME (MINUTES) 600 Figure 28: VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA concentration-time curves in plasma following 150 mg/kg i .p . administration of each compound to rats (n=8/time point, error bars=S.D.). Table 9: The time to peak ( t m a x ) and peak concentration ( C m a x ) i n t i s s u e s and plasma f o l l o w i n g VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n . tmax ( m i ") Cmax (ug/g or mL, S.D.) VPA 2-ene 2 , 3 ' - d i e n e VPA 2-ene 2 , 3 ' - d i e n e PLASMA 30 30 15 142.7(51.8) 143.0(50.0) 168.2(13.9) LIVER 30 30 45 2 2 5 . 6 ( 6 3 . l ) a 2 4 4 . 5 ( 6 4 . 9 ) b 76.1(19.0) KIDNEY 45 30 60 1 7 9 . 6 ( 3 2 . 0 ) a 99.5(29.5) 75.9(6.7) HEART 45 30 45 52.7(13.3) 66.3(29.8) 42.0(12.6) LUNG 45 45 45 9 3 . 2 ( 1 3 . 0 ) ° 5 2 . 8 ( 1 7 . 8 ) b 80.8(15.7) WHOLE BRAIN 30 30 45 50.6(13.0) 68.8(19.8) 56.2(8.4) S i g n i f i c a n t d i f f e r e n c e determined by one-way ANOVA and Newman-Keuls Test (a) s i g n i f i c a n c e between VPA and 2 , 3 ' - d i e n e (p<0.05) (b) s i g n i f i c a n c e between 2-ene and 2 , 3 ' - d i e n e (p<0.05) (c) s i g n i f i c a n c e between VPA and 2-ene (p<0.05) Table 10: AUC 0 to 10 h i n t i s s u e s f o l l o w i n g i . p . a d m i n i s t r a t i o n of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA. A U C 0 - 1 0 h ( " 9 ' n / 9 or mL, S.D.) VPA 2-ene 2 , 3 ' - d i e n e PLASMA 455 (68) 497 ( 3 8 ) a 401 (46) LIVER 854 (124) b 384 (63) 133 (15) KIDNEY 698 (114)b 273 (41) 121 (30) HEART 138 ( 2 5 ) b 79 (16) 52 (8) LUNG 221 ( 3 9 ) c d 147 (18) 135 (26) WHOLE BRAIN 105 ( 2 3 ) c d 48 (10) 56 (10) S i g n i f i c a n t d i f f e r e n c e determined by one-way ANOVA and Newman-Keuls Test (a) s i g n i f i c a n c e between 2-ene and 2 , 3 ' - d i e n e (p<0.05) (b) s i g n i f i c a n c e between VPA, 2-ene, and 2 , 3 ' - d i e n e (p<0.05) (c) s i g n i f i c a n c e between VPA and 2-ene (p<0.05) (d) s i g n i f i c a n c e between VPA and 2 , 3 ' - d i e n e (p<0.05) r a t e ( f i g u r e 29) . S i m i l a r r e s u l t s were observed i n the kidneys, h e a r t , and lungs ( f i g u r e s 30 - 3 2 ) . Following the equivalent s i n g l e dose a d m i n i s t r a t i o n of each compound, the peak concentration of ( E , E ) - 2 , 3 ' - d i e n e VPA a t t a i n e d i n the l i v e r was s i g n i f i c a n t l y l e s s (p<0.05) than both VPA and (E)-2-ene VPA ( t a b l e 9 ) . The times required to reach peak l i v e r concentrations f o r each compound were s i m i l a r . A comparison of the AUCo-ioh values i n l i v e r f o r each compound showed that the value f o r ( E , E ) - 2 , 3 ' - d i e n e VPA was s i g n i f i c a n t l y l e s s (p<0.05) than that of VPA and (E)-2-ene VPA (table 10). S i m i l a r trends were found i n the kidneys, h e a r t , and lungs ( t a b l e s 9 and 10). The A U C o - i o h values f o r VPA i n the various peripheral t i s s u e s i n d i c a t e d that a l a r g e p o r t i o n of the dose could be accounted f o r i n the l i v e r f r a c t i o n whereas d i s t r i b u t i o n of VPA i n t o the heart and lungs were somewhat s m a l l e r . The d i s t r i b u t i o n of (E)-2-ene VPA i n t o the various p e r i p h e r a l t i s s u e s f o l l o w i n g s i n g l e dose a d m i n i s t r a t i o n i n decreasing order were the l i v e r , kidneys, l u n g s , and heart . For ( E , E ) - 2 , 3 ' - d i e n e VPA, the d i s t r i b u t i o n i n t o the l i v e r , kidneys, and lungs appeared to be equivalent whereas the heart received a smaller f r a c t i o n of the dose. 3 . 6 . 3 P r o f i l e s i n Brain The concentration-time p l o t s f o r VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA were compared i n whole brain and no apparent d i f f e r e n c e s i n the e l i m i n a t i o n p r o f i l e s were observed f o r each compound ( f i g u r e 3 3 ) . S i m i l a r r e s u l t s were observed from the concentration-time p l o t s f o r each compound i n the various pooled brain sections (appendices 1 - 9 ) . 100 1000.0 - j - # VPA o (E ) -2 -ENEVPA • (E,E)—2,3'—DIENE VPA O O S _L • 0.1 — ' -I J 1.0E-2 H 1 1 1 0 200 400 600 TIME (MINUTES) Figure 29: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n l i v e r f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 101 1000.0 - T -100.0 CD CD Z5 o h -< 10.0 — LU O z o o 1.0 0.1 • VPA o (E)-2-ENEVPA • (E,E)-2,3'—DIENE VPA 0 200 400 TIME (MINUTES) 600 Figure 30: VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA concentration-time curves in kidneys following 150 mg/kg i .p . administration of each compound to rats (n=8/time point, error bars=S.D.). 102 Figure 31: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n heart f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 103 Figure 32: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n lungs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . 100.0 10.0 cn Z5 o h -< Or: 1.0 O z o o 0.1 1.0E-2 • VPA o (E ) -2 -ENEVPA E,E)-2,3'-DIENE VPA 1 o 0 200 400 600 TIME (MINUTES) Figure 33: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA concentration-time curves i n whole b r a i n f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (n=8/time p o i n t , e r r o r bars=S.D.) . In the whole brain f r a c t i o n and the various i n d i v i d u a l pooled b r a i n s e c t i o n s the t m a x and C m a x values f o r VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA were compared (table 11). No s i g n i f i c a n t d i f f e r e n c e s were observed i n the C m a x values of these compounds i n the whole b r a i n . Nor were there s i g n i f i c a n t d i f f e r e n c e s found i n the C m a x values between VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n the i n d i v i d u a l b r a i n r e g i o n s . The A U C o - i o h value f o r VPA i n whole brain was s i g n i f i c a n t l y greater than e i t h e r (E)-2-ene VPA or ( E , E ) - 2 , 3 ' - d i e n e VPA. The k i n e t i c p r o f i l e s and the AUCo-ioh values from the i n d i v i d u a l pooled brain regions were s i m i l a r to those i n whole brain ( t a b l e 12) and hence, the d i s t r i b u t i o n of VPA throughout the brain appeared homogeneous. S i m i l a r r e s u l t s were observed f o r (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. 3.7 TISSUE DISTRIBUTION AND KINETIC PROFILES OF METABOLITES OF VPA 3 . 7 . 1 P r o f i l e s of VPA Metabolites i n Plasma The k i n e t i c p r o f i l e of VPA and i t s biotransformation products were monitored over a 10 hour period i n plasma. For ease of presentation the metabolites were categorized i n t o unsaturated and p o l a r compounds. Following VPA a d m i n i s t r a t i o n , seven unsaturated metabolites were detected i n plasma ( f i g u r e 34) . The major unsaturated metabolites were (E)-2-ene VPA, 3-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA (concentration exceeding 1.0 ug/mL). Minor metabolites were (Z)-2-ene VPA, ( E , Z ) - 2 , 3 ' -diene VPA, ( E ) - 2 , 4 - d i e n e VPA, and 4-ene VPA. The r a t e of d e c l i n e f o r the unsaturated metabolites appeared to be s i m i l a r to that of the parent Table 11: The time to peak ( t m a x ) and peak concentration ( C m a x ) i n brain t i s s u e s f o l l o w i n g e i t h e r VPA, (E)-2-ene VPA, or ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n . tmax (min) C m a x (ug/g,S.D.) VPA 2-ene 2 . 3 ' - d i e n e VPA 2-ene 2 , 3 ' - d i e n e WHOLE BRAIN 30 30 45 50.6(13.0) 68.8(19.8) 56.2(8.4) HIP 30 30 30 45.4 48.7 40.5 SC 15 30 60 51.2 47.3 28.2 IC 30 30 60 52.4 52.8 28.6 CER 15 30 60 52.1 30.0 57.3 OLF 30 30 15 55.3 49.7 31.6 CC 15 15 15 69.3 37.4 39.7 SN 15 30 15 58.7 49.4 31.7 MED 15 15 30 46.2 31.9 27.6 CP 30 30 15 45.1 50.5 34.6 Table 12: AUC from 0 to 10 hours i n the i n d i v i d u a l pooled b r a i n s e c t i o n s f o l l o w i n g i . p . a d m i n i s t r a t i o n of e i t h e r VPA, (E)-2-ene VPA, or ( E , E ) - 2 , 3 ' - d i e n e VPA. AUCo-ioh (ug-h/g) VPA 2-ene 2 , 3 ' - d i e n e WHOLE BRAIN 105 ( 2 3 ) a b 48 (10) 56 (10) HIP 106.4 44.2 39.9 SC 134.4 51.0 36.6 IC 114.6 48.5 44.2 CER 119.6 47.5 55.6 OLF 104.6 54.4 45.0 CC 118.0 43.2 42.7 SN 123.6 51.3 44.4 MED 132.6 51.3 46.3 CP 106.8 47.1 52.7 S i g n i f i c a n t d i f f e r e n c e determined by one-way ANOVA and Newman-Keuls Test (a) s i g n i f i c a n c e between VPA and 2-ene VPA (p<0.05) (b) s i g n i f i c a n c e between VPA and 2 , 3 ' - d i e n e VPA (p<0.05) 108 ,E)-2,3'-DIENE VPA .Z)-2,3'-DIENE VPA 1000.0 - r -• VPA • ( E • (E v(E)-2,4-DIENE VPA o ( E ) - 2 - E N E VPA • ( Z j - 2 - E N E VPA A 3 - E N E VPA A 4 - E N E VPA O O 1.0E-3 1.0E-5 0 200 400 600 TIME (MINUTES) Figure 34: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . compound. The plateau phase associated with the plasma d e c l i n e of VPA was a l s o apparent f o r the m e t a b o l i t e s . The major polar metabolites observed i n plasma were 5-OH VPA and 3-keto VPA with 2-PGA, 4-keto VPA, 3-OH VPA, and 4-OH VPA present i n l e s s e r amounts ( f i g u r e 3 5 ) . The e l i m i n a t i o n p r o f i l e s of these p o l a r metabolites appeared to correspond to the d e c l i n e of the parent compound from plasma. 3.7.2 P r o f i l e s of VPA Metabolites i n Peripheral Tissues In the l i v e r , f i v e unsaturated metabolites were detected ( f i g u r e 36) of which two, (E)-2-ene VPA and 3-ene VPA, were major metabolites w h i l e (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA, and 4-ene VPA represented the minor m e t a b o l i t e s . The major p o l a r metabolites observed i n l i v e r f o l l o w i n g VPA a d m i n i s t r a t i o n were 2-PGA, 5-OH VPA, and the 0 - o x i d a t i o n products 3-keto VPA and 3-OH VPA. The minor p o l a r metabolites were 4-keto VPA and 4-OH VPA ( f i g u r e 37). The p r o f i l e of the unsaturated metabolites of VPA observed i n kidneys, h e a r t , and lungs were s i m i l a r to those found i n l i v e r (appendices 10 - 12). However, not present i n l i v e r but quantitated i n kidneys and lungs was ( E ) - 2 , 4 - d i e n e VPA. The concentration of ( E ) - 2 , 4 -diene VPA i n these t i s s u e s was s i m i l a r to that of ( E , E ) - 2 , 3 ' - d i e n e VPA. The p o l a r metabolites observed i n the kidneys, h e a r t , and lungs f o l l o w i n g VPA a d m i n i s t r a t i o n were s i m i l a r to those found i n l i v e r (appendices 13 - 15). 110 CD =5 o r— < LU O Z o o 1000.0 -r-100.0 10.0 1.0 4 s 0.1 4 -1.0E-2 1.0E-3 • VPA H 4-KETO VPA v 3-KETO VPA O 3 - 0 H VPA A 4 -OH VPA A 5 -OH VPA • 2-PGA 0 200 400 600 TIME (MINUTES) Figure 35: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 1000.0 100.0 - -10.0 1.0 - -0.1 1.0E-2 - -1.0E-3 1.0E-4 • VPA n(E,E)-2,3'-DIENE VPA • (E 1Z)-2,3'-DIENE VPA o ( E ) - 2 - E N E VPA A 3 —ENE VPA A4 -ENE VPA oo - A — —o—' • c n 0 1 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . • VPA • 4-KETO VPA v3-KETO VPA O 3 - 0 H VPA A 4-OH VPA A 5 - O H VPA • 2-PGA 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 3 . 7 . 3 P r o f i l e s of VPA Metabolites i n Brain Following VPA i . p . a d m i n i s t r a t i o n , the q u a n t i t a t i o n of metabolites i n the i n d i v i d u a l remaining whole b r a i n f r a c t i o n was not p o s s i b l e . Of the several metabolites detected the s i g n a l s corresponding to these metabolites were below the l i m i t s of the assay and hence, only the parent compound could be quantitated (appendix 16). Because of the small s e c t i o n s of r a t brain being analyzed, pooling of i n d i v i d u a l regions from the 8 r a t s was necessary f o r the d e t e c t i o n of m e t a b o l i t e s . An area i n the brain considered to be high i n GABA a c t i v i t y i s the s u b s t a n t i a n i g r a . The unsaturated metabolites observed i n the s u b s t a n t i a n i g r a f o l l o w i n g VPA a d m i n i s t r a t i o n were 3-ene VPA and (E)-2-ene VPA, each at concentrations l e s s than 0.1 ug/g, ( E , Z ) - 2 , 3 ' - d i e n e VPA at approximately 10 ng/g, and 4-ene VPA at 0.1 ng/g ( f i g u r e 3 8 ) . At no time was the major plasma m e t a b o l i t e , ( E , E ) - 2 , 3 ' - d i e n e VPA, detected i n t h i s region of the b r a i n . The p o l a r metabolites detected i n the s u b s t a n t i a n i g r a , i n descending amounts, were 5-OH VPA, 4-OH VPA, 3-keto VPA, 2-PGA, 4-keto VPA, and 3-OH VPA ( f i g u r e 3 9 ) . The concentrations of the p o l a r metabolites were s i m i l a r but l e s s than 1 ug/mL. The other pooled b r a i n s e c t i o n s assayed were the hippocampus, superior c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , cerebellum, o l f a c t o r y b u l b , corpus c a l l o s u m , medulla, and caudate putamen. The nonpolar and p o l a r metabolites observed i n these areas were s i m i l a r to those found i n the s u b s t a n t i a n i g r a (appendices 17 - 32) . 100.0 10.0 — 1.0 — 1.0E-2 1.0E-3 — 1.0E-4 1.0E-5 • VPA o ( E ) - 2 - E N E VPA A 3-ENE VPA A 4 -ENE VPA • (E,Z)-2,3'-DIENE VPA o . i -4- ga O . . ^ f \ A ' - A O v. A \ 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled/time p o i n t ) . 115 100.0 cn Z5 O < or: LU o z o o • VPA • 4-K V3 - K O 3 - 0 • 2-PGA ETO VPA ETO VPA V V . VPA PA PA 10.0 1.0 0.1 1.0E-2 0 200 400 TIME (MINUTES) 600 Figure 39: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled/time p o i n t ) . 3 . 8 TISSUE DISTRIBUTION AND KINETIC PROFILES OF METABOLITES OF (E)-2-ENE VPA 3 . 8 . 1 P r o f i l e s of (E)-2-ene VPA Metabolites i n Plasma The k i n e t i c p r o f i l e s of (E)-2-ene VPA and i t s biotransformed products were determined i n plasma f o l l o w i n g the i . p . a d m i n i s t r a t i o n of a dose of (E)-2-ene VPA containing 5% (Z)-2-ene VPA. The metabolites monitored by GC-MS were the same as those assayed a f t e r VPA a d m i n i s t r a t i o n . The (Z)-2-ene VPA quantitated i n plasma and t i s s u e s f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n was not considered a metabolite but instead as a by-product of (E)-2-ene VPA s y n t h e s i s . The major nonpolar metabolites found i n plasma were 3-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA and VPA ( f i g u r e 4 0 ) . The minor unsaturated products (<1 ug/mL) detected were ( E , Z ) - 2 , 3 ' - d i e n e VPA, ( E ) - 2 , 4 - d i e n e VPA, and 4-ene VPA. Of the several polar metabolites detected i n plasma, 3-keto VPA was the major product while 3-OH VPA, 4-0H VPA, 5-OH VPA and 2-PGA were present i n l e s s e r amounts ( f i g u r e 41). The e l i m i n a t i o n p r o f i l e i n plasma of the unsaturated and polar metabolites of (E)-2-ene VPA resembled the e l i m i n a t i o n p r o f i l e of the parent compound. 3 . 8 . 2 P r o f i l e s of (E)-2-ene VPA Metabolites i n Peripheral Tissues The major unsaturated metabolite quantitated i n the l i v e r f r a c t i o n a f t e r (E)-2-ene VPA a d m i n i s t r a t i o n was 3-ene VPA ( f i g u r e 4 2 ) . Somewhat s u r p r i s i n g l y VPA, a product of (E)-2-ene VPA r e d u c t i o n , was the main metabolite i n l i v e r . In f a c t , the concentration of VPA a t t a i n e d l e v e l s greater than that of the parent compound at 6 hours f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n . Thus, reduction of (E)-2-ene VPA appears to be a 117 1000.0 CD Z 3 o < on Ld o z o o 100.0 10.0 1.0 0.1 - -1.0E-2 O (E ) -2 -ENEVPA v (Z ) -2 -ENEVPA • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA • 2,4-DIENE VPA A 3-ENE VPA A 4 -ENE VPA • VPA 0 200 400 600 TIME (MINUTES) Figure 40: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 118 1000.0 13 o o z o o 100.0 10.0 p 1.0 < LY. 0.1 1.0E-2 1.0E-3 o (E ) -2 -ENEVPA V (Z ) -2 -ENE VPA • 3-KETO VPA • 3-OH VPA A 4-OH VPA A 5-OH VPA • 2-PGA 0 200 400 600 TIME (MINUTES) Figure 41: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 119 1000.0 - r -cn Z5 o < o z o o 100.0 o ~~o oo 10.0 1.0 ~ 0.1 1.0E-2 O ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3-ENE VPA A 4 -ENE VPA • VPA 0 200 400 600 TIME (MINUTES) Figure 42: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . prominent metabolic pathway f o r t h i s compound. Other minor unsaturated metabolites of (E)-2-ene VPA i n l i v e r were (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA, and 4-ene VPA. The major p o l a r metabolites observed f o l l o w i n g (E)-2- ene VPA a d m i n i s t r a t i o n were 3-keto VPA and 3-OH VPA, both of which are metabolic products of mitochondrial /J-oxidation ( f i g u r e 4 3 ) . Minor polar metabolites were 4-OH VPA, 5-OH VPA, and 2-PGA. The r e s u l t s observed i n the kidneys, h e a r t , and lungs were s i m i l a r to that i n the l i v e r (appendices 33 - 3 8 ) . In c o n t r a s t to the r e s u l t s from l i v e r was the absence of ( E ) - 2 , 4 - d i e n e VPA, and the concentration of both 3-ene VPA and VPA i n these peripheral t i s s u e s was approximately 1 0 - f o l d l e s s . For the p o l a r m e t a b o l i t e s , 4-OH VPA was not detected i n the k i d n e y s , h e a r t , and lungs but was found i n the l i v e r . 3.8.3 P r o f i l e s of (E)-2-ene VPA Metabolites i n B r a i n The unsaturated metabolites detected i n the remaining whole brain f r a c t i o n f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n , i n order of decreasing b r a i n c o n c e n t r a t i o n , were 3-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and (E,Z)-2 , 3 ' - d i e n e VPA (appendix 3 9 ) . VPA was present at l e v e l s equivalent to 3-ene VPA. The two p o l a r products of (E)-2-ene VPA metabolism observed i n the remaining whole b r a i n were the /3-oxidation products 3-keto VPA and 3- OH VPA a l s o present i n plasma (appendix 40) . Polar products a r i s i n g from microsomal metabolism such as 4-OH VPA and 5-OH VPA were below the l e v e l s of d e t e c t i o n . The metabolites of (E)-2-ene VPA were also quantitated i n the hippocampus, superior c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , cerebellum, o l f a c t o r y b u l b s , corpus c a l l o s u m , s u b s t a n t i a n i g r a , medulla, and the 121 1000.0 - r -100.0 Z5 o f— < LU o z o o 10.0 1.0 0.1 o ( E ) - 2 - E N E VPA v ( Z ) - 2 - E N E V P A T 3 -KETO VPA • 3 - O H VPA A 4 - O H VPA A 5 - O H VPA • 2 - P G A 1 .0E-2 - | 1 1 1 0 200 400 600 TIME (MINUTES) Figure 43: Concentration-time p l o t s of (E)-2-ene VPA and i t s polar metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . caudate putamen. The nonpolar metabolites of (E)-2-ene VPA observed i n the s u b s t a n t i a n i g r a i n order of decreasing b r a i n concentration were 3-ene VPA, ( E , E ) - , and ( E , Z ) - 2 , 3 ' - d i e n e VPA ( f i g u r e 44). VPA was present as the reduced product of (E)-2-ene VPA at l e v e l s s i m i l a r to that of 3-ene VPA. The p o l a r metabolites observed were the ^ - o x i d a t i o n products, 3-keto VPA and 3-OH VPA, and 4- and 5-OH VPA ( f i g u r e 45) . The concentrations of the p o l a r metabolites i n the s u b s t a n t i a n i g r a were s i m i l a r to each other and were quantitated at submicrogram l e v e l s . The unsaturated and p o l a r metabolites observed i n the other regions of the brain f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n were comparable to the r e s u l t s found i n the s u b s t a n t i a n i g r a (appendices 41 -5 6 ) . 3 . 9 TISSUE DISTRIBUTION AND KINETIC PROFILES OF METABOLITES OF (E,E)-2,3'-DIENE VPA 3 . 9 . 1 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Plasma The metabolites of ( E , E ) - 2 , 3 ' - d i e n e were quantitated i n plasma over a 10 hour p e r i o d . The metabolites chosen f o r q u a n t i t a t i o n were those that appear as products of VPA metabolism. The ( E , Z ) - 2 , 3 ' - d i e n e VPA was not considered to be a metabolite but a by-product of (E ,E)-2 , 3 ' - d i e n e VPA s y n t h e s i s , since the dose of ( E , E ) - 2 , 3 ' - d i e n e VPA contained 3 - 5% of the (E ,Z)-isomer. The metabolites observed i n plasma f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , i n order of decreasing abundance, were 3-ene VPA, (E)-2-ene VPA, and a d i e n e , and VPA ( f i g u r e 46) . At 10 hours f o l l o w i n g (E ,E)-CD CD Z5 o !< on h-z LU o z o o 100.0 10.0 1.0 o— A . X 0.1 — \ \ \ \ N " A _ 1 -o (E ) -2 -ENEVPA v ( Z J - 2 - E N E V P A • (E,E)-2 I 3 , -DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3 -ENEVPA • VPA -o _ _ ' —• •— — — A 1.0E-2 /\ I • 1.0E-3 0 200 400 TIME (MINUTES) 600 Figure 44: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8 pooled/time p o i n t ) . CD CD Z5 o !< (Y L U O Z o o 100.0 O ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • 3-KETO VPA • 3-OH VPA A 4 -OH VPA A 5-OH VPA 10.0 1.0 0.1 1.0E-2 - -1.0E-3 0 200 400 TIME (MINUTES) 600 Figure 45: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA (containing 5% (Z)-2-ene VPA) to r a t s (n=8 pooled/time p o i n t ) . 125 1000.0 — i — M (E,E)-2 ,3 ' -DIENE VPA • (E,Z)—2,3'—DIENE VPA A DIENE o (E ) -2 -ENE VPA A 3 - E N E VPA • VPA 100.0 — cn Z5 O r— < LY. I— Z LU O z o o 10.0 - -1.0 0.1 1.0E-2 0 200 400 TIME (MINUTES) 600 Figure 46: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E,E)-2,3 '-diene VPA ( c o n t a i n i n g 5% (E,E)-2,3 '-diene VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , the concentration of (E)-2-ene VPA and 3-ene VPA i n plasma exceeded that of the parent compound, suggesting that the h a l f - l i v e s of these metabolites were greater than that of the parent compound. Only three p o l a r metabolites were detected i n plasma. The most abundant was 3-keto VPA, with smaller amounts of 3-OH VPA and 5-OH VPA a l s o o c c u r r i n g ( f i g u r e 47). 3 . 9 . 2 P r o f i l e s of ( E , E ) -2 ,3 ' - d i e n e VPA Metabolites i n Peripheral Tissues Most notable i n the l i v e r f r a c t i o n f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , were the high l e v e l s of VPA, (E)-2-ene VPA, and 3-ene VPA r e s p e c t i v e l y . A l l three of the reduced products of ( E , E ) - 2 , 3 ' - d i e n e VPA metabolism exceeded the l e v e l of the parent compound w i t h i n 200 minutes of a d m i n i s t r a t i o n , suggesting a high turnover r a t e of t h i s diene via reduction ( f i g u r e 48). As i n the case of plasma, p o l a r metabolites found i n l i v e r were 3-keto VPA and 3-OH VPA with 5-OH VPA present i n minor q u a n t i t i e s ( f i g u r e 49) . The concentration of the p o l a r metabolites surpassed the l e v e l of the parent compound w i t h i n 240 minutes of a d m i n i s t r a t i o n . The metabolites observed i n the kidneys, h e a r t , and lungs f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n were s i m i l a r to those found i n l i v e r although i n l e s s e r q u a n t i t i e s (appendices 57 - 5 9 ) . The concentration of the metabolites d i d not at anytime exceed the l e v e l of the parent compound during the 10 hour p e r i o d . The p o l a r metabolites detected i n the kidneys, h e a r t , and lungs were comparable to the metabolites found i n the l i v e r (appendices 60 - 62) . The d e c l i n e of these p o l a r products from the kidneys, h e a r t , and lungs appeared to 127 1000.0 E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA • 3-KETO VPA • 3-OH VPA v 5-OH VPA 100.0 Cn Z5 o on LU o z o o 10.0 1.0 0.1 1.0E-2 0 200 400 TIME (MINUTES) 600 Figure 47: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n plasma f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% (E,Z)-2»3 '-diene VPA) to r a t s (n=8/time p o i n t , S.D. omitted for c l a r i t y ) . 128 100.0 • (E,E)-2,3 ,-DIENE VPA • (E >Z)-2,3 ,-DIENE VPA A DIENE o (E ) -2 -ENEVPA A 3-ENE VPA • VPA 10.0 CD CD Z 3 o !< cr LU o z o o 1.0 0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Figure 48: Concentration-time plots of (E,E)-2,3'-diene VPA, i t s unsaturated metabolites, and VPA in l i v e r following the administration of 150 mg/kg i.p. of (E,E)-2,3'-diene VPA (containing 3-5% (E,Z)-2,3'-diene VPA) to rats (n=8/time point, S.D. omitted for c l a r i t y ) . 129 100.0 10.0 -4-cn CD Z5 o 1.0 4-< or: LU o z o o 0.1 -f-• (E,E)-2,3'-DIENE VPA • ( E,z)—2 , 3 ' —DIENE VPA • 3-KETO VPA • 3-OH VPA v 5-OH VPA 1.0E-2 - h 1.0E-3 0 200 400 600 TIME (MINUTES) Figure 49: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n l i v e r f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . p a r a l l e l the clearance of the parent compound. Although i n some i n s t a n c e s , as i n the case of the heart t i s s u e , clearance of ( E , E ) - 2 , 3 ' -diene VPA was f a s t e r than the polar m e t a b o l i t e s . The r e s u l t was the concentrations of the polar metabolites exceeded the parent compound approximately 300 minutes f o l l o w i n g a d m i n i s t r a t i o n . 3 . 9 . 3 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n B r a i n VPA was the major metabolite observed i n the remaining whole brain f r a c t i o n f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n ( f i g u r e 50). The concentration of VPA i n the brain exceeded the l e v e l of the parent ( E , E ) - 2 , 3 ' - d i e n e VPA compound w i t h i n 200 minutes a f t e r a d m i n i s t r a t i o n . The unsaturated metabolites detected i n the remaining whole b r a i n were 3-ene VPA, (E)-2-ene VPA, and another d i e n e , although a l l were present i n l e s s e r amounts r e l a t i v e to VPA. The p o l a r metabolites observed i n the remaining whole b r a i n were the /3-oxidation products of VPA, namely 3-keto VPA and 3-0H VPA (appendix 6 3 ) . The metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA were a l s o quantitated i n the various pooled b r a i n s e c t i o n s . As i n the case of the remaining whole b r a i n , the VPA concentration i n the s u b s t a n t i a n i g r a , an area of high GABA a c t i v i t y , exceeded that of ( E , E ) - 2 , 3 ' - d i e n e VPA a f t e r 350 minutes ( f i g u r e 51). The unsaturated metabolites observed i n decreasing concentrations were 3-ene VPA, (E)-2-ene VPA, and a d i e n e . The e l i m i n a t i o n of the unsaturated metabolites appeared to be s i m i l a r to that of ( E , E ) - 2 , 3 ' - d i e n e VPA. The polar metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA detected i n the s u b s t a n t i a n i g r a were the /J-oxidation products of VPA and 5-0H VPA ( f i g u r e 52). The e l i m i n a t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA from the s u b s t a n t i a n i g r a appeared to be greater than the hydroxyl 131 100.0 - r • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA A DIENE O (E ) -2 -ENE VPA A 3-ENE VPA • VPA 10.0 cn cn Z3 o !< on Ld o z o o 1.0 0.1 1.0E-2 + 1.0E-3 0 200 400 600 TIME (MINUTES) Figure 50: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n whole b r a i n f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 132 • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3 ' -DIENE VPA A DIENE O (E ) -2 -ENE VPA A 3-ENE VPA • VPA 0 200 400 600 TIME (MINUTES) Figure 51: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E ,E)-2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8 pooled/time p o i n t ) . 133 1.0E-3 4 1 1 1 0 200 400 600 TIME (MINUTES) Figure 52: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n s u b s t a n t i a n i g r a f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA (containing 3-5% ( E , Z ) - 2 , 3 ' - d i e n e VPA) to r a t s (n=8 pooled/time p o i n t ) . metabolites as the concentration of these polar compounds exceeded that of the parent at approximately 400 minutes. The metabolites observed in the other regions of the brain following single dose administration of (E,E)-2,3'-diene VPA were si m i l a r to those found in the substantia nigra (appendices 64 - 79). As in peripheral tissues, the major metabolite observed in the brain was VPA. 3.10 PLASMA PROTEIN BINDING OF (E,E)-2,3'-DIENE VPA AND (E,Z)-2,3'-DIENE VPA When the elimination characteristics of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA were compared in plasma, the clearance of the diene from plasma appeared to be fastest. Similar results were found for the peripheral tissues and brain. It was thought that variations in plasma protein binding between (E,E)-2,3'-diene VPA and VPA or (E)-2-ene VPA might explain these differences in tissue clearance. Knowing the degree of plasma protein binding of the isomers (E,Z)- and (E,E)-2,3'-diene VPA might also explain the presence of only the (E,Z)-isomer in the brain following VPA administration. While the plasma protein binding c h a r a c t e r i s t i c s of VPA and (E)-2-ene VPA in rats i s known, the binding of (E,E)-2,3'-diene VPA has not yet been established. Therefore, binding of (E,Z)- and (E,E)-2,3'-diene VPA was determined from the collected rat plasma samples. Following the i.p. administration of (E,E)-2,3'-diene VPA containing 3 - 5% of the (E,Z)-isomer, plasma protein binding was determined by u l t r a c e n t r i f u g a t i o n from plasma samples c o l l e c t e d over a 10 hour p e r i o d . For the various t o t a l concentrations of (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA measured i n plasma over t i m e , a corresponding free plasma concentration was determined. From t h i s d a t a , the degree of plasma p r o t e i n binding of both (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA was c a l c u l a t e d (table 13). At an equivalent t o t a l plasma concentration of approximately 12 - 14 ug/mL, 96% of ( E , E ) - 2 , 3 ' - d i e n e VPA was bound to plasma p r o t e i n compared to 35% f o r the ( E , Z ) - i s o m e r . The binding isotherm f o r ( E , E ) - 2 , 3 ' - d i e n e VPA and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n d i c a t e d that the binding of each compound was l i n e a r over the concentration ranges studied ( f i g u r e 53). 3.11 ANTICONVULSANT EVALUATION OF VPA, (EJ-2-ENE VPA, (E,E)-2,3'-DIENE VPA, AND (E,Z)-2,3'-DIENE VPA IN RATS 3.11.1 PTZ-Induced Seizure Test Upon the s . c . a d m i n i s t r a t i o n of 75 mg/kg of PTZ to eight c o n t r o l r a t s , a l l animals e x h i b i t e d c l o n i c seizures w i t h i n 30 minutes, the considered endpoint. The percentage of r a t s protected against PTZ-induced c l o n i c s e i z u r e s by e i t h e r VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, or ( E , Z ) - 2 , 3 ' - d i e n e VPA at various doses are summarized i n t a b l e 14. The ( E , E ) - 2 , 3 ' - d i e n e VPA at 200 mg/kg was found to be s i g n i f i c a n t l y l e s s a c t i v e (p<0.05) as an anticonvulsant agent when compared to VPA. In f a c t , the dose-response curves ( f i g u r e 54) d e p i c t s ( E , E ) - 2 , 3 ' - d i e n e VPA as being l e s s potent and l e s s e f f e c t i v e than VPA, (E)-2-ene VPA, or ( E , Z ) - 2 , 3 ' - d i e n e VPA. Q u a n t i t a t i o n of anticonvulsant a c t i v i t y Table 13: Plasma p r o t e i n binding of ( E , E ) - 2 , 3 ' - d i e n e VPA and ( E , Z ) - 2 , 3 ' -diene VPA i n r a t plasma by u l t r a c e n t i f u g a t i o n ; n=8 ( S . D . ) -( E , E ) - 2 , 3 ' - d i e n e VPA Total Concentration (ug/mL) 168.2 (36.6) 140.5 (13.9) 151.4 (19.7) 154.9 (25.6) 62.6 (17.8) 26.3 (4.5) 13.5 (3.1) ( E , Z ) - 2 , 3 ' - d i e n e VPA Total Concentration (ug/mL) 11.6 (3.1) 9.6 (0.7) 10.6 (1.6) 10.3 (1.3) 3 . 9 (1.0) 1.2 (0.4) 1.2 (0.2) Free Concentration (ug/mL) 46.5 (19.4) 32.5 (6.7) 32.3 (6.0) 29.8 (4.9) 10.7 (5.3) 0 . 8 (0.3) 0 . 8 (0.1) Free Concentration (ug/mL) 7.4 (2.8) 6.3 (1.5) 6 . 6 (1.1) 6.8 (1.3) 3.0 (1.1) 0 . 8 (0.3) 0.3 (0.1) Percent Bound 74.2 (6.4) 76.7 (4.4) 78.7 (2.7) 80.5 (3.1) 85.1 (3.4) 97.1 (0.4) 95.2 (1.0) Percent Bound 34.9 (7.1) 33.7 (5.5) 38.1 (5.9) 34.4 (7.31) 28.7 (4.1) 43.2 (10.4) 75.8 (6.3) 137 TOTAL ( E , E ) - 2 , 3 , - d i e n e VPA CONCENTRATION (ug /mL) Figure 53: Binding isotherm of ( E , E ) - 2 , 3 ' - d i e n e VPA and ( E , Z ) - 2 , 3 ' -diene VPA i n r a t plasma as determined by u l t r a c e n t r i f u g a t i o n (n=8). Table 14: The percent of r a t s protected from PTZ-induced seizures a f t e r the i . p . a d m i n i s t r a t i o n of 70 mg/kg s . c . of PTZ 30 minutes post drug (n=8/dose). Dose (mg/kg) 30 75 150 200 300 400 VPA 12.5% 25% 37.5% 87.5% 100% -(E,E)-2,3'-DIENE VPA - 12.5% 12.5% * 25% 62.5% 75% (E,Z)-2,3'-DIENE VPA - 12.5% 12.5% 50% 100% -(E)-2-ENE VPA - 0% 25% 62.5% 100% (*) s i g n i f i c a n t compared to VPA (p<0.05) CO CO 0 . 1 0 0 1 . 0 0 0 DOSE (mmol /kg) Figure 54: Dose-response curves f o r VPA, (E)-2-ene VPA, (E,Z)-2,3'-diene VPA, and (E,E)-2,3 ' -diene VPA following 150 mg/kg i .p . administration of each compound to rats (n=8/point). CO l O from the dose-response curves demonstrated t h a t , based on the ED50 v a l u e s , ( E , E ) - 2 , 3 ' - d i e n e VPA was s i g n i f i c a n t l y l e s s potent (p<0.05) when compared to VPA ( t a b l e 15). The ED50 values f o r (E)-2-ene VPA and (E ,Z)-2 , 3 ' - d i e n e VPA d i d not d i f f e r s i g n i f i c a n t l y from that of VPA. 3.11 .2 Observed Adverse E f f e c t s of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and ( E , Z ) - 2 , 3 ' - d i e n e VPA The i . p . a d m i n i s t r a t i o n of 30 - 150 mg/kg of VPA d i d not produce any v i s i b l e adverse e f f e c t s , whereas doses of 200 and 300 mg/kg d i d e l i c i t a s l i g h t sedative e f f e c t that was seen 10 minutes f o l l o w i n g a d m i n i s t r a t i o n . This was c h a r a c t e r i z e d by a quiet h y p n o t i c - l i k e s t a t e but without the l o s s of r i g h t i n g . Within an hour t h i s VPA induced sedation had subsided and the animal appeared to regain movement. Upon (E)-2-ene VPA a d m i n i s t r a t i o n at doses of 75 - 300 mg/kg, a marked sedative e f f e c t was noted which was c h a r a c t e r i z e d by a reduction i n body movement and the l o s s of r i g h t i n g w i t h i n 2 - 3 minutes. At 10 minutes p o s t - i n j e c t i o n , the animal appeared to be i n a h y p n o t i c - l i k e motionless s t a t e which l a s t e d f o r 2 - 3 hours. Rats administered 75 - 150 mg/kg of ( E , E ) - 2 , 3 ' - d i e n e VPA d i s p l a y e d moderate sedation w i t h i n 17 minutes, while doses of 200 - 400 mg/kg e l i c i t e d a moderate to heavy sedative e f f e c t w i t h i n 10 minutes of a d m i n i s t r a t i o n . In a d d i t i o n to the sedative/hypnotic e f f e c t , a s t i f f n e s s or an increase i n muscle tone of the hindlegs was noted f o l l o w i n g doses of 150 and 200 mg/kg. As the dose was increased to 300 and 400 mg/kg, whole body r i g i d i t y was observed. The t o n i c i t y of the muscles was severe enough to almost mask the s e i z u r e a c t i v i t y induced by PTZ. This apparent neurotoxic e f f e c t of ( E , E ) - 2 , 3 ' - d i e n e VPA was not permanent as the r a t s Table 15: The mean e f f e c t i v e doses against PTZ-induced seizures and the slopes of the l o g dose-response p l o t s f o r each compound tested i n r a t s COMPOUND VPA ( E , Z ) - 2 , 3 ' - d i e n e VPA (E)-2-ene VPA ( E , E ) - 2 , 3 ' d i e n e VPA () - 95% confidence l i m i t * - s i g n i f i c a n t (p<0.05) ED50 (mmol/kg) 1 .1(1 .0-1.3) 1 .2(1.1-1.4) 1 .3(1.1-1.4) 1 . 9 ( 1 . 4 - 2 . 5 ) * Slope 1 .2(1.1-1.3) 1 . 2 ( 0 . 8 - 1 . 8 ) 1 .2(1 .0-1 .3) 1 .7(1 .1-2.5) appeared to returned to t h e i r normal s t a t e of a c t i v i t y w i t h i n 4 hours of i n j e c t i o n . The a d m i n i s t r a t i o n of ( E , Z ) - 2 , 3 ' - d i e n e VPA at doses of 150 - 300 mg/kg r e s u l t e d i n a mild sedative e f f e c t s i m i l a r to that seen f o r VPA. No l o s s of r i g h t i n g o c c u r r e d . Absent was the muscle r i g i d i t y and/or p a r a l y s i s associated with the (E ,E)-isomer. 4 . DISCUSSION 4.1 CHEMICAL SYNTHESES The f o l l o w i n g compounds were s u c c e s s f u l l y synthesized using methods that maximized both y i e l d and s t r u c t u r a l s e l e c t i v i t y . Previous methods used f o r the synthesis of VPA metabolites s u f f e r e d from e i t h e r low y i e l d s or poor isomeric s e l e c t i v i t y . Because several grams of each substance under study were needed to perform both pharmacokinetic and anticonvulsant a c t i v i t y s t u d i e s , m o d i f i c a t i o n of reported syntheses was undertaken to circumvent these l i m i t a t i o n s . 4 . 4 . 1 2 - n - P r o p y l - ( E ) - 2 - P e n t e n o i c A c i d The synthesis of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d by the method of Acheampong (1985) r e s u l t e d i n low y i e l d s (<10%) and the presence of a s i g n i f i c a n t quantity of 2 - n - p r o p y l - ( Z ) - p e n t e n o i c a c i d . The synthesis required bromination of VPA i n the a - p o s i t i o n followed by e s t e r i f i c a t i o n of the c a r b o x y l i c a c i d moiety. Upon i s o l a t i o n , the compound was debrominated and then r a p i d l y d i s t i l l e d to y i e l d the ethyl e s t e r of 2-n-propyl -2-pentenoic a c i d . L i b e r a t i o n of the e s t e r by a l k a l i n e h y d r o l y s i s followed by d i s t i l l a t i o n afforded a mixture that c o n s i s t e d of 80% 2 - n -propyl - ( E ) - 2 - p e n t e n o i c a c i d and 20% the (Z)-isomer. Because the synthesis of gram q u a n t i t i e s of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d i n an i s o m e r i c a l l y pure form using t h i s method would prove too l a b o r i o u s and i n e f f i c i e n t , an a l t e r n a t e method was i n v e s t i g a t e d . Syntheses of a , ^ - u n s a t u r a t e d e s t e r s v i a the r e g i o s e l e c t i v e a d d i t i o n of e l e c t r o p h i l e s to e s t e r enolates have i n the past proved to be s u c c e s s f u l ( P f e f f e r and S i l b e r t , 1971; Rathke and S u l l i v a n , 1972; P f e f f e r et al., 1973; Kajikawa et al., 1975; Kende and Toder, 1982). Based on t h i s methodology, an attempt was made to synthesize 2 - n - p r o p y l -(E)-2-pentenoic a c i d by f i r s t r e a c t i n g ethyl v a l e r a t e with propionaldehyde i n an a l d o l condensation r e a c t i o n . The r e s u l t i n g /J-hydroxysaturated e s t e r was then mesylated with methanesulfonyl c h l o r i d e . N u c l e o p h i l i c e l i m i n a t i o n of the mesylate moiety with potassium hydride was thought to give r i s e to an a , ^ - u n s a t u r a t e d e s t e r ; however, upon GC-MS a n a l y s i s of the crude product, no t r a c e of the unsaturated e s t e r was present. The r e a c t i o n was repeated several times with each attempt producing s i m i l a r r e s u l t s . In a r e c e n t l y published method f o r the synthesis of 2 - n - p r o p y l -(E)-2-pentenoic a c i d , t o l u e n e s u l f o n y l c h l o r i d e was used to form a t o s y l e s t e r of ethyl /3-hydroxypentanoate (Rettenmeier et al., 1989). N u c l e o p h i l i c e l i m i n a t i o n of the t o s y l group by 1 ,8-d i a z a b i c y c l o [ 5 . 4 . 0 ] u n d e c - 7 - e n e (DBU) afforded the a,/3-unsaturated e s t e r i n q u a n t i t a t i v e y i e l d s . The f i n a l product c o n s i s t e d of 90% of the (E)-isomer. A r e a c t i o n time of 72 hours f o r the formation of the t o s y l e s t e r i n combination with d i f f i c u l t i e s i n i s o l a t i n g the t o s y l a t e d product from the solvent made t h i s method l e s s amenable f o r l a r g e s c a l e synthesis required f o r our r e s e a r c h . Since the r a t e l i m i t i n g step f o r the synthesis of 2 - n - p r o p y l - ( E ) -2-pentenoic a c i d by the method of Rettenmeier et al. (1989) was the formation of the t o s y l a t e , use of a mesylate would s i g n i f i c a n t l y reduce the r e a c t i o n time. The formation of the mesyl e s t e r from the /J-hydroxyunsaturated e s t e r was completed w i t h i n an hour i n q u a n t i t a t i v e y i e l d s and with l i t t l e or no side product contamination. Since the r e a c t i o n was c a r r i e d out i n THF, i s o l a t i o n of the product by f l a s h evaporation followed by d i s t i l l a t i o n proved to be an e f f i c i e n t recovery method. N u c l e o p h i l i c e l i m i n a t i o n of the mesylate moiety by r e f l u x i n g with DBU followed by s a p o n i f i c a t i o n of the ethyl e s t e r afforded a product that was 95% i s o m e r i c a l l y pure. From the NMR spectrum ( f i g u r e 6 ) , the product was determined to have the ( E ) - c o n f i g u r a t i o n based on the s i g n a l f o r the C3 - p r o t o n . Depending on the c o n f i g u r a t i o n of the double bond at A S the proton at C3 may be s u s c e p t i b l e to the e l e c t r o n i c e f f e c t s of the TT e l e c t r o n of the carbonyl moiety ( f i g u r e 5 5 ) . For 2 - n - p r o p y l - ( E ) -2 - p e n t e n o i c a c i d , the proton at C3 i s c l o s e to the carbonyl group thereby i n t e r a c t i n g with the 7T e l e c t r o n s , 7 . e . deshielded and hence, the s i g n a l f o r the proton occurs downfield at 6 . 8 ppm. For 2 - n - p r o p y l - ( Z ) -2 - p e n t e n o i c a c i d , the C3 - p r o t o n i s away or shielded from the 7r e l e c t r o n s r e s u l t i n g i n an u p f i e l d s i g n a l at 5 . 9 ppm. Since the integrated s i g n a l at 6 . 8 ppm was greater than the s i g n a l at 5 . 9 ppm by nearly 2 0 - f o l d , i t was concluded that the majority of synthesized product was 2 - n - p r o p y l - ( E ) -2 - p e n t e n o i c a c i d . Therefore, the synthesis of 2 - n - p r o p y l - ( E ) -2 - p e n t e n o i c a c i d by the n u c l e o p h i l i c e l i m i n a t i o n of the mesyl e s t e r by DBU proved to be both s t e r e o s e l e c t i v e and h i g h l y e f f i c i e n t f o r our purposes. 4 . 1 . 2 2 - ( ( Z ) - T - P r o p e n y l ) - ( E ) - 2 - P e n t e n o i c A c i d The synthesis of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) -2 - p e n t e n o i c a c i d was s i m i l a r to that of 2 - n - p r o p y l - ( E ) -2 - p e n t e n o i c a c i d . The e l e c t r o p h i l i c a d d i t i o n of propionaldehyde to an cr,/3-unsaturated e s t e r enolate afforded 146 2 - n - P R 0 P Y L - ( E ) - 2 - P E N T E N 0 I C A C I D 2 - n - P R 0 P Y L - ( Z ) - 2 - P E N T E N 0 I C A C I D Figure 55: Chemical s t r u c t u r e s of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d and 2 - n - p r o p y l - ( Z ) - 2 - p e n t e n o i c a c i d i l l u s t r a t i n g the s h i e l d i n g and d e s h i e l d i n g e f f e c t s of the v i n y l i c protons. a fi-hydroxy-ft' ,7'- u n s a t u r a t e d e s t e r . The c o n f i g u r a t i o n of the double bond at A^ w i l l depend on the c o n f i g u r a t i o n of the i n i t i a l r e a c t a n t . In other words, to synthesize the (Z)-isomer the s t a r t i n g a,/J-alkenyl e s t e r must possess the ( E ) - c o n f i g u r a t i o n p r i o r to geometric i n v e r s i o n (Kende and Toder, 1982). To synthesize ethyl 2 - ( l ' - h y d r o x y p r o p y l ) - ( Z ) - 3 -pentenoate, the s t a r t i n g compound was (E)-2-pentenoic a c i d . Upon a l k y l a t i n g the s t a r t i n g reactant with propionaldehyde, the double bond of the r e s u l t i n g hydroxy unsaturated e s t e r was s h i f t e d to the 3 - p o s i t i o n by geometric i n v e r s i o n to the ( Z ) - c o n f i g u r a t i o n (scheme 2 ) . Following mesylation of the hydroxyl group and n u c l e o p h i l i c e l i m i n a t i o n with DBU, s a p o n i f i c a t i o n of the product gave good y i e l d s of 2 - ( ( Z ) - l ' - p r o p e n y l ) -(E)-2-pentenoic a c i d . The c o n f i g u r a t i o n of the product was r e a d i l y determined by NMR. S i m i l a r s h i e l d i n g and d e s h i e l d i n g e f f e c t s ( f i g u r e 56) akin to 2 - n -propyl - ( E ) - 2 - p e n t e n o i c a c i d were used to d i s t i n g u i s h the p o s i t i o n a l isomers. The c o n f i g u r a t i o n at A 2 can also be r a t i o n a l i z e d by the behavior of the C3-proton s i m i l a r to that of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d . The proton at C3 corresponds to a s i g n a l at 6 . 8 - 6.95 ppm when A2 possesses the ( E ) - c o n f i g u r a t i o n ( f i g u r e 56). The absence of a s i g n a l at 5.92 ppm ( f i g u r e 5 5 ) , corresponding to the shielded C3~proton i n the ( Z ) - c o n f i g u r a t i o n , i n d i c a t e s that the r e a c t i o n was s t e r e o s p e c i f i c f o r the (E)-isomer at A 2 . The proton at C 2 ' a l s o produces a c h a r a c t e r i s t i c s i g n a l that d i f f e r e n t i a t e s between the (E)- and ( Z ) - c o n f i g u r a t i o n at A 1 . The s i g n a l at 5.8 ppm corresponds to the shielded C 2 ' - p r o t o n when AA has the (Z)-c o n f i g u r a t i o n ( f i g u r e 56). However, the same proton when A 1 ' possesses the ( E ) - c o n f i g u r a t i o n r e s u l t s i n a s i g n a l at 6.1 ppm. From the NMR 148 6.8 ppm 6.1 ppm 6.95 ppm 2- ( (E) -1 ' - P R 0 P E N Y L ) - ( E ) - 2 - P E N T E N 0 I C ACID 2-((Z)-1 ' - P R 0 P E N Y L ) - ( E ) - 2 - P E N T E N 0 I C ACID Figure 56: Chemical s t r u c t u r e s of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d and 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d i l l u s t r a t i n g the s h i e l d i n g and d e s h i e l d i n g e f f e c t s of the v i n y l i c protons. spectrum ( f i g u r e 8 ) , the i n t e n s i t y of the integrated s i g n a l at 5 . 8 ppm was 95% greater than the s i g n a l at 6.1 ppm. Therefore, the m a j o r i t y of the product was comprised of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d with a small f r a c t i o n being 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . Although 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was p r e v i o u s l y i d e n t i f i e d as a contaminant of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d synthesis (Acheampong and Abbott, 1985; Lee et al., 1989), i t had not p r e v i o u s l y been s t e r e o s e l e c t i v e l y synthesized as a major product. Therefore, the DBU e l i m i n a t i o n r e a c t i o n of a mesylate intermediate proved to be an e f f i c i e n t synthesis f o r the d e s i r e d product. 4 . 1 . 3 2 - ( ( E ) - l ' - P r o p e n y l ) - ( E ) - 2 - P e n t e n o i c Acid The synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was f i r s t described by Acheampong and Abbott (1985). The method was not s t e r e o s e l e c t i v e , since 3 to 4 isomeric dienoates were detected upon GC-MS a n a l y s i s . In a d d i t i o n , the y i e l d was low. Therefore, attempts were made to improve both the y i e l d and the isomeric p u r i t y of t h i s product by modifying the reported s y n t h e s i s . The method used f o r the synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d was s i m i l a r to that of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d . Since the c o n f i g u r a t i o n of the s t a r t i n g e s t e r d i c t a t e s the geometry of the f i n a l product, to synthesize 2 - ( ( E ) - l ' - p r o p e n y l ) -(E)-2-pentenoic a c i d , the r e a c t i n g e s t e r must be ethyl (Z)-2-pentenoate (scheme 3 ) . Synthesis of t h i s e s t e r required a c a t a l y s t , 18-crown-6. In the previous method f o r the synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d by Acheampong and Abbott (1985), a c a t a l y s t was not employed which probably explains the poor y i e l d s achieved. Mesylation of the hydroxy substituent followed by n u c l e o p h i l i c e l i m i n a t i o n with potassium hydride afforded mainly the e t h y l 2 - ( ( E ) - 1 ' -propenyl)-(E)-2-pentenoate. Potassium hydride was i n i t i a l l y used f o r the e l i m i n a t i o n r e a c t i o n u n t i l the u t i l i t y of DBU was d i s c o v e r e d . When KH was used, the p u r i t y of the e s t e r product obtained depended on r e a c t i o n c o n d i t i o n s . With c a r e f u l c o n t r o l of the r e a c t i o n temperature and t i m e s , together with the use of r e - d i s t i l l e d reagents, isomeric p u r i t y was achieved. Following s a p o n i f i c a t i o n of the e s t e r , f r a c t i o n a l d i s t i l l a t i o n gave a product that was 95% 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d with a of 46% y i e l d . The stereochemistry and p u r i t y of the product was determined by NMR. The s i g n a l at 6 . 8 ppm corresponded to the deshielded proton at C3 when A 2 has the ( E ) - c o n f i g u r a t i o n ( f i g u r e 56). In c o n t r a s t to the NMR r e s u l t s of 2 - ( ( Z ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d , the v i n y l i c proton s i g n a l at 6.1 ppm was approximately 2 0 - f o l d greater than the s i g n a l at 5.8 ppm ( f i g u r e 10). Therefore, the f i n a l product c o n s i s t e d mostly of 2-( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . The synthesis of 2 - ( ( E ) - l ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was a s i g n i f i c a n t improvement over the reported method of Acheampong and Abbott (1985). The isomeric p u r i t y of the f i n a l product was 95 - 97% as determined by both GC-MS and NMR. In a d d i t i o n , the y i e l d was s u b s t a n t i a l l y increased thereby allowing the e f f i c i e n t synthesis of the diene f o r pharmacological study. 4 . 2 ASSAY DEVELOPMENT FOR VPA AND ITS METABOLITES IN RAT TISSUE Successful GC-MS assays of VPA metabolites i n various b i o l o g i c a l f l u i d s have been reported f o r the ethyl (Granneman et al., 1984), TMS (Nau et al., 1981; Tatsuhara et al., 1987; Rettenmeier et al., 1989), or t-BDMS (Acheampong et al., 1984; Abbott et al., 1986) d e r i v a t i v e s . Of the three d e r i v a t i v e s , the greatest number of VPA metabolites separated w i t h i n the s h o r t e s t period of time during a s i n g l e a n a l y s i s was the t -BDMS e s t e r s . In a d d i t i o n , the r e l a t i v e s e n s i t i v i t y f o r " VPA and unsaturated metabolites using the t-BDMS d e r i v a t i v e was 2 0 - f o l d greater than those observed f o r the TMS d e r i v a t i v e (Abbott et al., 1986). Two major drawbacks prevented use of the t-BDMS d e r i v a t i v e f o r the a n a l y s i s of VPA and i t s metabolites i n r a t t i s s u e samples obtained from the current study. A n a l y s i s of t-BDMS d e r i v a t i v e s by s e l e c t e d ion monitoring under EI c o n d i t i o n s has a reported l i m i t of d e t e c t i o n of 0.1 ug/mL ( s i g n a l - t o - n o i s e <3) (Abbott et al., 1986). In a d d i t i o n , the assay procedure f o r VPA and VPA metabolites using t-BDMS d e r i v a t i v e s required a 1 mL t i s s u e sample. Since i n d i v i d u a l pooled r a t b r a i n s e c t i o n s were to be assayed, the amount of b r a i n t i s s u e obtained would not allow f o r sample volumes of that s i z e . I f l e s s than 1 mL of sample were to be assayed, the concentration of some VPA metabolites would f a l l below the l i m i t of d e t e c t i o n of the assay. The second problem of the reported assay was the r e s o l u t i o n of diene m e t a b o l i t e s . As t-BDMS e s t e r s , r e s o l u t i o n of ( E , Z ) - 2 , 3 ' - d i e n e VPA and ( E ) - 2 , 4 - d i e n e VPA was not p o s s i b l e ( f i g u r e 57). Previous studies that have quantitated VPA metabolites i n the l i v e r (Nau and Loscher, 1985) and b r a i n t i s s u e (Loscher and Nau, 1982) have measured only 7 fej 2 B JU 1+4 TIME (MINUTES) gure 57: Mass chromatogram at m/z 139 of t-BDMS d e r i v a t i v e s of (A) peak l = ( E , Z ) - 2 , 3 ' - d i e n e VPA and peak 2 = ( E , E ) - 2 , 3 ' - d i e n e VPA i n c o n t r o l human serum; (B) peak 3=(Z)-2,4-diene VPA and peak 4=(E)-2,4-diene VPA i n c o n t r o l human serum; (C) serum sample of a p a t i e n t on VPA therapy c o n t a i n i n g peaks 1 to 4, on an 0V-1701 column. metabolites i n the brain and 4 i n the l i v e r . The diene detected i n the b r a i n was 2 , 3 ' - d i e n e VPA with the stereochemistry not r e p o r t e d . The GC-MS assay employed TMS d e r i v a t i v e s and while not known f o r c e r t a i n , our experience i n d i c a t e d the method was l i k e l y not capable of separating the diene isomers. Thus, an a l t e r n a t i v e assay method having adequate s e n s i t i v i t y to detect t r a c e amounts of VPA metabolites from small sample s i z e s and the a b i l i t y to resolve the diene metabolites was r e q u i r e d . A negative ion GC-MS assay was therefore i n v e s t i g a t e d f o r our purposes. A recent negative ion chemical i o n i z a t i o n (NICI) method developed f o r the d e t e c t i o n of VPA and VPA metabolites i n human serum samples using a pentafluorobenzyl (PFB) d e r i v a t i v e proved to be h i g h l y s e n s i t i v e and s e l e c t i v e f o r the diene metabolites (Kassahun et a 7 . , 1989). The l i m i t of d e t e c t i o n f o r VPA based on a 200 uL serum sample was 2 ng/mL ( s i g n a l - t o - n o i s e >3) (Kassahun et a / . , 1990). Moreover, the s e n s i t i v i t y obtained with PFB d e r i v a t i v e s of VPA and i t s metabolites under NICI c o n d i t i o n s by s e l e c t e d ion monitoring was 30 - 50 times greater than that of t h e i r corresponding t-BDMS d e r i v a t i v e s by EI GC-MS. In a d d i t i o n , separation of the diene m e t a b o l i t e s , ( E , Z ) - 2 , 3 ' - d i e n e VPA and ( E ) - 2 , 4 -diene VPA, was a l s o p o s s i b l e by NICI GC-MS ( f i g u r e 5 8 ) . Therefore, with t h i s method appearing to meet the needs of our study, procedures f o r the a n a l y s i s of VPA and VPA metabolites i n r a t t i s s u e were d e v i s e d . In c o n t r a s t to the simple e x t r a c t i o n of a c i d i f i e d serum or urine (Abbott et a / . , 1986; Kassahun et a 7 . , 1989), the i s o l a t i o n of VPA and i t s metabolites from r a t t i s s u e homogenates required s p e c i f i c c o n s i d e r a t i o n s . Because extracted t i s s u e homogenates contained s i g n i f i c a n t l y more endogenous material than from e i t h e r urine or serum samples, i n t e r f e r e n c e with the d e t e c t i o n of the VPA metabolites TIME (MINUTES) Figure 58: Mass chromatogram at m/z 139 of PFB d e r i v a t i v e s of (A) peak l = ( E , Z ) - 2 , 3 ' - d i e n e VPA and peak 2 = ( E , E ) - 2 , 3 ' - d i e n e VPA i n c o n t r o l human serum; (B) peak 3=(Z)-2,4-diene VPA and peak 4=(E)-2,4-diene VPA i n c o n t r o l human serum; (C) serum sample of a p a t i e n t on VPA therapy containing peaks 1 to 4 , on a DB-1 column. 155 o c c u r r e d . To remedy t h i s problem a "clean-up" procedure was employed that c o n s i s t e d of a back e x t r a c t i o n step f o r the removal of neutral compounds ( f i g u r e 59). In a d d i t i o n , a 5 m pre-column was attached to the DB-1 chromatographic column to r e t a i n low v o l a t i l e compounds that would otherwise accumulate on the a n a l y t i c a l column. When the r e s o l u t i o n f o r some VPA metabolites began to d e t e r i o r a t e , a 0 . 5 m p o r t i o n of the pre-column at the i n l e t end was removed thereby r e s t o r i n g r e s o l u t i o n ( f i g u r e 6 0 ) . Approximately 43 extracted and d e r i v a t i z e d samples were analyzed before column performance s i g n i f i c a n t l y d e t e r i o r a t e d and the pre-column m o d i f i c a t i o n was r e q u i r e d . The NICI GC-MS method f o r the a n a l y s i s of VPA and VPA metabolites was not without i t s drawbacks. Because of the nature of the sample analyzed and the method used f o r producing negative i o n s , the i n t e g r i t y of the source appeared to be compromised over a r e l a t i v e l y short period of t i m e . Frequent source c l e a n i n g was required every 300 samples, thus making t h i s method somewhat l e s s amenable f o r routine a n a l y s i s . However, since no other method achieved the s e n s i t i v i t y and separation c a p a b i l i t i e s needed f o r t h i s study, r e g u l a r maintenance of the source was an accepted inconvenience. A second disadvantage, unrelated to i n s t r u m e n t a t i o n , was the u n a v a i l a b i l i t y of s p e c i f i c deuterium l a b e l l e d VPA metabolites f o r use as i n t e r n a l standards. Deuterated analogues, s i m i l a r to those used f o r VPA, (E)-2-ene VPA, or 3-keto VPA q u a n t i t a t i o n , were necessary i n order to account f o r the p h y s i c a l or chemical changes c h a r a c t e r i s t i c of some i n d i v i d u a l metabolites during e x t r a c t i o n . F a i l u r e to account f o r these p a r t i c u l a r changes may r e s u l t i n greater v a r i a t i o n s i n the assay. Metabolites that may r e q u i r e the use of these deuterated analogues are 156 1 00 uL distilled water 50 uL internal standard s 55 uL of 1 N HCl Extracted with 2 x 500 uL of ethyl acetate Discard aqueous fraction Extract organic fraction with 400 uL of 1 N NaOH 1 Discard organic fraction Add 85 ul_ of 4 N HCl to aqueous fraction Extract with 1 mL of ethyl acetate j Dry organic fraction over sodium sulfate Reduce volume to 1 00 uL under nitrogen 1 0 uL of diisopropylethylamine 1 0 uL of quinol (1 mg/mL in ethyl acetate) 1 0 uL of PFB (30'/. in ethyl acetate) Heat 45 min at 6 0 ° C 50 uL of MSTFA Heat 45 min at 6 0 ° C I Inject 1 uL Figure 59: General e x t r a c t i o n and d e r i v a t i z a t i o n scheme f o r the NICI GC-MS a n a l y s i s of VPA and VPA metabolites i n r a t plasma and homogenized t i s s u e . 157 Figure 60: Mass chromatogram at m/z 139 of ( E , Z ) - 2 , 3 ' - d i e n e VPA ( a ) , ( E ) - 2 , 4 - d i e n e VPA ( b ) , and ( E , E ) - 2 , 3 ' - d i e n e VPA (c) in l i v e r homogenate. Mass chromatogram one was a obtained from a d e t e r i o r a t e d DB-1 column f i t t e d with a 5 m pre-column w h i l e mass chromatogram two was obtained f o l l o w i n g the removal of a 0 . 5 m p o r t i o n of the pre-column. the diene m e t a b o l i t e s , the hydroxy m e t a b o l i t e s , and d i c a r b o x y l i c a c i d m e t a b o l i t e s . In s p i t e of minor problems, the NICI GC-MS assay provided picogram or b e t t e r s e n s i t i v i t y . A l a r g e number of VPA metabolites could be q u a n t i t a t e d w i t h i n a reasonably short period of time. 4 . 3 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF VPA IN RATS The pharmacokinetics of VPA i n plasma has been thoroughly c h a r a c t e r i z e d i n the r a t f o l l o w i n g i . p . and i . v . doses of 15 - 600 mg/kg (Dickinson et a 7 . , 1979; Loscher et a 7 . , 1988). D i s t r i b u t i o n studies of VPA i n p e r i p h e r a l t i s s u e s and brain have also been performed (Aly and A b d e l - L a t i f , 1980; Nau and Loscher, 1982; Hariton et a 7 . , 1984). The present study of the pharmacokinetics and t i s s u e d i s t r i b u t i o n of VPA was undertaken to take advantage of our improved metabolite assay, and to provide v a l i d comparison f o r s i m i l a r studies of s e l e c t e d m e t a b o l i t e s . The d i s t r i b u t i o n and pharmacokinetics of pharmacologically a c t i v e VPA metabolites were to be determined f o r several major organs, i n c l u d i n g various b r a i n s e c t i o n s of r a t s and compared to VPA. 4 . 3 . 1 P r o f i l e of VPA i n Plasma Following a s i n g l e dose of 150 mg/kg i . p . of VPA, peak plasma concentrations of 142.7 ± 51.8 ug/mL were attained w i t h i n 30 minutes, thus absorption of the drug i n t o the general c i r c u l a t i o n was not r a p i d . S i m i l a r r e s u l t s or f i n d i n g s have been reported (Hariton et a 7 . , 1984; Morton, 1984). The e l i m i n a t i o n phase of VPA i n plasma d i s p l a y e d a t r a n s i e n t increase i n the VPA concentration at 240 minutes f o l l o w i n g drug a d m i n i s t r a t i o n . S i m i l a r r e s u l t s f o r VPA were found i n r a t s f o l l o w i n g e i t h e r i . p . or i . v . a d m i n i s t r a t i o n (Dickinson et a 7 . , 1979; Lawyer et a 7 . , 1980; Ogiso et a 7 . , 1986). It appears that extensive enterohepatic r e c y c l i n g of VPA was responsible f o r the t r a n s i e n t increase i n plasma VPA concentration during the e l i m i n a t i o n phase. B i l e e x t e r i o r i z e d r a t s d i d not e x h i b i t t h i s secondary increase i n VPA plasma concentration but gave the t y p i c a l l o g - l i n e a r d e c l i n e (Dickinson et al., 1979). It can be s a f e l y assumed that the observed secondary increase i n VPA plasma concentration was a r e s u l t of h e p a t o b i l i a r y r e c y c l i n g . The n o n - l i n e a r i t y of VPA e l i m i n a t i o n from plasma does not allow f o r the determination of an apparent e l i m i n a t i o n r a t e constant by conventional pharmacokinetic techniques. The use of a complex m u l t i -compartmental model i s required but the present data were not s u f f i c i e n t f o r such r i g o r o u s modelling techniques. Clearance of VPA from plasma was c a l c u l a t e d from the A U C o - i o h value and expressed as C l o - i O h A since b i o a v a i l a b i l i t y (F) f o r VPA i n r a t s was not determined. B i o a v a i l a b i l i t y f o r VPA i n man and animals a f t e r o r a l a d m i n i s t r a t i o n i s c l o s e to 1 (Loiseau et a 7 . , 1975; Meinardi et a 7 . , 1975; Schobben et a 7 . , 1975). In a d d i t i o n , the l i t e r a t u r e value f o r A U C o - i o h i n r a t s f o l l o w i n g i . v . a d m i n i s t r a t i o n of 150 mg/kg of VPA was approximately 372 ug'h/mL (Dickinson et a 7 . , 1979). Since t h i s value i s comparable to the 455 ± 68 ug*h/mL obtained f o l l o w i n g i . p . a d m i n i s t r a t i o n i n t h i s study, F can be assumed to equal one. Clearance from 0 - 1 0 hours f o r VPA i n plasma was then c a l c u l a t e d to be 92 ± 14 mL/h. Previous studies r e p o r t i n g pharmacokinetic parameters f o r VPA were determined from time zero to i n f i n i t y . The current data were obtained between zero and ten hours as the observed enterohepatic r e c y c l i n g of VPA prevented the estimation of a h a l f - l i f e v a l u e . In order to compare our r e s u l t s with that of l i t e r a t u r e v a l u e s , a h a l f - l i f e value f o r VPA from previous studies i n r a t s , administered a s i m i l a r dose, was used to c a l c u l a t e the r e s i d u a l d e c l i n e of VPA a f t e r 10 hours f o l l o w i n g drug a d m i n i s t r a t i o n . The h a l f - l i f e value of VPA i n the r a t was approximately 15 minutes (Dickinson et al., 1979; Hariton et al., 1984; Ogiso et al., 1986). An apparent e l i m i n a t i o n r a t e constant was then c a l c u l a t e d and used f o r the estimation of AUC and Cl to time i n f i n i t y . C a l c u l a t e d AUC and Cl values from time zero to i n f i n i t y f o l l o w i n g a s i n g l e dose i . p . a d m i n i s t r a t i o n of 150 mg/kg of VPA were 460 ± 68 ug'h/mL and 5 ± 1 mL/min/kg, r e s p e c t i v e l y . From the clearance v a l u e , a volume of d i s t r i b u t i o n (V<j) of 119 ± 18 mL/kg was determined. These pharmacokinetic parameters i n r a t were comparable to those i n the l i t e r a t u r e . Values f o r Cl and Vj were 4.17 mL/min/kg (Loscher, 1978) and 143.0 ± 7 mL/kg (Ogiso et al., 1986), r e s p e c t i v e l y . 4 . 3 . 2 P r o f i l e of VPA i n Peripheral Tissues The a n a l y s i s of VPA i n peripheral t i s s u e s f o l l o w i n g drug a d m i n i s t r a t i o n has p r e v i o u s l y been l i m i t e d to r a t l i v e r and kidneys and was based on the d i s t r i b u t i o n of a r a d i o l a b e l e d analog of VPA (Aly and A b d e l - L a t i f , 1980). Thus, i t was f e l t that a comprehensive study of the t i s s u e d i s t r i b u t i o n and k i n e t i c s of VPA would provide useful information f o r the comparison of VPA with other metabolites to be s t u d i e d . D i s t r i b u t i o n of VPA i n t o the l i v e r , kidneys, h e a r t , and lungs was found to reach peak drug concentrations w i t h i n 30 - 45 minutes. The t i s s u e p r o f i l e s were very s i m i l a r to that i n plasma, i n c l u d i n g the secondary increase i n VPA concentration due to enteroheptic r e c y c l i n g . Thus, the l i v e r , kidneys, h e a r t , and lungs along with plasma can be considered as a homogeneous u n i t g e n e r a l l y termed the c e n t r a l compartment. The t m a x values f o r VPA i n the peripheral t i s s u e s and i n plasma were s i m i l a r (table 9 ) . A f t e r i . p . a d m i n i s t r a t i o n , the d i s t r i b u t i o n of [^C]VPA i n r a t s gave maximum r a d i o a c t i v i t y i n the l i v e r w i t h i n 30 minutes, which corresponded to peak plasma concentrations (Aly and A b d e l - L a t i f , 1980). S i m i l a r l y , f o l l o w i n g o r a l a d m i n i s t r a t i o n of VPA i n the mouse, peak concentrations of VPA i n plasma corresponded to peak concentrations i n the l i v e r (Nau and Loscher, 1985). While 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 p r o f i l e s of VPA i n the p e r i p h e r a l t i s s u e s were very s i m i l a r , the concentrations were not. The concentration of VPA i n l i v e r was the h i g h e s t , followed by kidneys, h e a r t , and l u n g s . In f a c t , the concentration of VPA i n l i v e r and kidneys was g r e a t e r than that i n plasma. Thus, tissue/plasma r a t i o s of VPA i n l i v e r and kidneys increased from 1.8 and 1.3 at 30 minutes to 4 . 6 and 1.8 at t i o h > r e s p e c t i v e l y ( f i g u r e 61). Liver/plasma r a t i o s i n mouse were reported to be i n the range of 1.5 - 3 .0 and i t was suggested that an a c t i v e t r a n s p o r t mechanism may be f u n c t i o n a l (Nau and Loscher, 1985). Such an assumption i s reasonable, since VPA at p h y s i o l o g i c a l pH i s more than 99% i o n i z e d and hence, would not g e n e r a l l y a t t a i n the high l e v e l s observed i n l i v e r by simple passive d i f f u s i o n (Frey and Loscher, 1978). However, s e l e c t i v e binding of VPA may a l s o be c o n t r i b u t i n g to the elevated r a t i o s observed i n l i v e r t i s s u e . Because the l i v e r and the 162 2.500 cn V P A ( E ) - 2 - E N E V P A ( E . E j - a ^ ' - D I E N E V P A 0 2.000 - t max < cn < 1.500 co _j CL Ld ZD CO CO 1.000-0.500--0.000 i WB I PLA LIV KID HEA LUN o i— < or CO CL Ld CO CO 5 - tioh 2--0 I WB PIA 1 LIV KID i HEA LUN Figure 61: Tissue/plasma r a t i o s of VPA, (E)-2-ene VPA, and (E ,E)-2 , 3 ' - d i e n e VPA i n whole b r a i n (WB), l i v e r (LIV) , kidneys (KID), heart (HEA), and lungs (LUN) of r a t s at t m a x and tioh f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound (n=8,error bars=S.D.) . kidneys are the major organs responsible f o r the e l i m i n a t i o n of VPA (Bruni and W i l d e r , 1979; Gugler and von Unruh, 1980; B a i l e r et al., 1985), i t would not be unusual to detect higher concentrations of drug i n these t i s s u e s . The high concentration of VPA i n l i v e r has been i m p l i c a t e d i n drug-induced l i v e r t o x i c i t y (Nau and Loscher, 1985). There appears to be much l e s s t i s s u e binding of VPA to heart and l u n g s . Tissue/plasma r a t i o s at t m a x and tjoh w e r e l e s s than one ( f i g u r e 61). The AUCo-ioh values f o r heart and lungs were 50% that of plasma (table 10). I t appears that the presence of VPA i n heart and lungs may be of l i t t l e c l i n i c a l s i g n i f i c a n c e . Metabolism of VPA by these organs appear to be minimal based on t h e i r metabolite p r o f i l e s being s i m i l a r to that of plasma. 4 . 3 . 3 P r o f i l e of VPA i n Brain The binding or accumulation of VPA i n brain could conceivably produce the " c a r r y - o v e r " e f f e c t of anticonvulsant a c t i v i t y that i s observed f o l l o w i n g the d i s c o n t i n u a t i o n of VPA. Results of the current study i n d i c a t e that VPA does not appear to bind or p e r s i s t r e l a t i v e to plasma i n whole brain t i s s u e f o l l o w i n g s i n g l e dose a d m i n i s t r a t i o n . The e l i m i n a t i o n of VPA was found to be r a p i d over the 10 hour period while the brain/plasma r a t i o s at t m a x and tioh were 0 . 3 and 0 . 0 0 1 , r e s p e c t i v e l y ( f i g u r e 61). S i m i l a r r e s u l t s were observed i n r a t s f o l l o w i n g constant rate a d m i n i s t r a t i o n of VPA f o r up to 14 days v i a an osmotic pump. Brain/plasma r a t i o s were i n the range of 0.03 - 0.2 (Loscher and Nau, 1983). Following an i . p . dose of 200 mg/kg of VPA i n mouse, the brain/plasma r a t i o was 0.2 at 30 minutes ( t m a x ) and then f e l l below the l i m i t of d e t e c t i o n at tsh (Nau and Loscher, 1982). The r a p i d clearance of VPA out of the b r a i n , which was demonstrated to be twice the r a t e of i t s e n t r y , i s l i k e l y associated with an a c t i v e transport mechanism such as the monocarboxylic a c i d c a r r i e r (Frey and Loscher, 1978; Cornford et a 7 . , 1985). Probenecid, a known i n h i b i t o r of the transport of monocarboxylic a c i d across the b l o o d - b r a i n b a r r i e r (Spector and Lorenzo, 1973; Pardridge and Oldendorf, 1977), can i n h i b i t the outward movement of VPA from the CNS. Therefore, concentrations of VPA i n the b r a i n may not be an appropriate i n d i c a t o r f o r p r e d i c t i n g anticonvulsant a c t i v i t y . Because the a n a l y s i s of VPA i n whole b r a i n does not provide information on the regional d i s t r i b u t i o n of the drug, various brain s e c t i o n s were assayed independently f o l l o w i n g VPA a d m i n i s t r a t i o n . The areas of b r a i n assayed were the hippocampus, s u p e r i o r c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , cerebellum, o l f a c t o r y b u l b , corpus c a l l o s u m , s u b s t a n t i a n i g r a , medulla, and the putamen. The b r a i n regions chosen f o r a n a l y s i s were based on s i m i l a r studies c a r r i e d out i n the r a t where these b r a i n s e c t i o n s were s e l e c t e d f o r t h e i r p o t e n t i a l GABA a c t i v i t y (Loscher and Nau, 1983; Hariton et a 7 . , 1984; Loscher et a 7 . , 1988). Of p a r t i c u l a r i n t e r e s t was the s u b s t a n t i a n i g r a , as t h i s i s a known area of high GABA a c t i v i t y (Iadorola and Gale, 1982). No regional d i f f e r e n c e s i n brain concentrations of VPA were found. The concentration of VPA i n the brain s e c t i o n s were s i m i l a r to that of whole b r a i n ( f i g u r e 62). The AUCo-ioh values f o r each b r a i n s e c t i o n analyzed d i d not d i f f e r from each other or from whole b r a i n . Tissue/plasma r a t i o s f o r the various areas of the b r a i n were i n the range of 0.3 - 0.4 at t m a x and 0.01 - 0.03 at tjoh ( f i g u r e 63). S i m i l a r r a t i o s were obtained from whole brain analyses. In a previous study 165 O Sc cn h-z LU O z o o 70 60 50 40 30 20 10-0-VPA L m a x ( E ) - 2 - E N E VPA (E.E)-2,3 ' -DIENE VPA 2 0 0 1 5 0 cn Z o < cn 1 0 0 H ; Ld O 5 0 O O CO 5 Q_ HIP SC IC CER OLF CC SN MED CP WB PLA 1.000 - r cn cn zs 0.750--O cn Ld o 0.500 g 0.250--0.000 tioh J2_ la. la . 0. 4 0 3 0 - - 1 0 0 HIP SC IC CER OLF CC SN MED CP WB PLA cn ZJ Z o cn + 2 0 H -z Ld O O < CO CL Figure 62: Concentrations of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n hippocampus (HIP), s u p e r i o r c o l l i c u l u s (SC), i n f e r i o r c o l l i c u l u s ( I C ) , cerebellum (CER), o l f a c t o r y bulbs (OLF), corpus callosum (CC), s u b s t a n t i a n i g r a (SN), medulla (MED), putamen (CP), whole b r a i n (WB), and plasma (PLA) of r a t s at t m a x and t ^ h f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound (WB&PLAn=8,HIP...CP pooled from 8 r a t s ) . 0.500 T max CZD VPA 1ZZ] ( E ) - 2 - E N ( E , E ) - 2 , 3 ' ENE VPA DIENE V P A O cr oo Q_ iii Z) 00 00 0.250 0.000 HIP SC IC CER OLF CC SN MED CP WB O 1.500 -r 1.250 t i o h cS 1.000 < ^ 0.750 D_ LU ZD 0.500 00 00 I— 0.250 0.000 _OJl / / / / rim I T > > / ' 1 r~,mm 1 A. HIP SC IC CER OLF CC SN MED CP WB Figure 63: Tissue/plasma ratios of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA in hippocampus (HIP), superior co l l icu lus (SC), infer ior col l iculus (IC), cerebellum (CER), olfactory bulbs (OLF), corpus callosum (CC), substantia nigra (SN), medulla (MED), putamen (CP), whole brain (WB), and plasma (PLA) of rats at t m a x and t j o h following 150 mg/kg i .p . administration of each compound (WB&PLA n=8,HIP...CP pooled from 8 rats) . f o l l o w i n g continuous a d m i n i s t r a t i o n of VPA to r a t s , comparable r e s u l t s were observed (Loscher and Nau, 1983). Concentrations of VPA i n b r a i n regions were found to be uniform and r e l a t i v e l y low compared to that i n plasma. Studies i n humans have shown that p a t i e n t s on VPA therapy had brain/plasma r a t i o s i n the range of 0.068 - 0.28 (Vajda et a l . , 1981). S i m i l a r l y , the CSF/plasma r a t i o s f o r VPA i n p a t i e n t s were i n the range 0.054 - 0.11 (Loscher et a 7 . , 1988). The appearance and d e c l i n e of VPA from whole brain and brain s e c t i o n s f o l l o w i n g drug a d m i n i s t r a t i o n to the r a t compared favorably with the r e s u l t s of previous s t u d i e s . The data can now be used with confidence to evaluate r e s u l t s obtained f o r VPA m e t a b o l i t e s . 4 . 4 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E)-2-ENE VPA IN RATS 4 . 4 . 1 P r o f i l e of (E)-2-ene VPA i n Plasma In humans and animals, (E)-2-ene VPA i s a major metabolite (Granneman et al., 1984; Abbott et a7, 1986; Kassahun et a 7 . , 1990) that has been shown i n rodents to possess anticonvulsant a c t i v i t y comparable to VPA (Loscher, 1981; Loscher and Nau, 1985; Abbott and Acheampong, 1988). Upon the d i s c o n t i n u a t i o n of VPA a d m i n i s t r a t i o n , (E)-2-ene VPA was s t i l l detected i n r a t b r a i n while VPA was absent from plasma (Loscher and Nau, 1983). These r e s u l t s l e d researchers to propose that the anticonvulsant a c t i v i t y observed post-VPA a d m i n i s t r a t i o n may be due to the presence i n b r a i n of (E)-2-ene VPA. Thus, the pharmacokinetic p r o f i l e of (E)-2-ene VPA was examined i n d e t a i l . 168 The (E)-2-ene VPA reached peak plasma concentrations of 143.0 ± 50.0 ug/mL w i t h i n 30 minutes f o l l o w i n g a d m i n i s t r a t i o n . Thereafter the plasma l e v e l of (E)-2-ene VPA began to d e c l i n e but was c h a r a c t e r i z e d 240 minutes l a t e r by a secondary increase i n (E)-2-ene VPA concentration s i m i l a r to that observed f o r VPA. H e p a t o b i l i a r y r e c y c l i n g of (E)-2-ene VPA would be the most l i k e l y explanation f o r t h i s increase i n (E)-2-ene VPA i n plasma c o n c e n t r a t i o n . In a previous study s i m i l a r e f f e c t s were observed i n r a t s f o l l o w i n g i . v . a d m i n i s t r a t i o n of (E)-2-ene VPA (Singh et a 7 . , 1990). This secondary increase i n (E)-2-ene VPA plasma l e v e l s was absent i n b i l e e x t e r i o r i z e d r a t s . Because an apparent e l i m i n a t i o n r a t e constant f o r (E)-2-ene VPA could not be obtained due to the n o n - l i n e a r i t y of the data from b i l i a r y e f f e c t s , a l i t e r a t u r e h a l f - l i f e value of 28 minutes was taken to c a l c u l a t e estimates of AUC and Cl/F from time zero to i n f i n i t y (Singh et al., 1990). C a l c u l a t e d values f o r AUC, C l / F , and V<j were estimated to be 518 ± 40 ug-h/mL, 5 ± 0.4 mL/min/kg, and 213 ± 25 mL/kg r e s p e c t i v e l y . These pharmacokinetic parameters f o r (E)-2-ene VPA i n plasma were comparable to l i t e r a t u r e values of 3 . 0 - 6.1 mL/min/kg and 230 - 240 mL/kg f o r Cl and V j , r e s p e c t i v e l y f o l l o w i n g i . v . a d m i n i s t r a t i o n of 20 -100 mg/kg of (E)-2-ene VPA (O'Connor et al., 1986; Singh et al., 1990). What was p a r t i c u l a r l y d i f f e r e n t about (E)-2-ene VPA was the k i n e t i c p r o f i l e of the terminal e l i m i n a t i o n phase. I t appeared that (E)-2-ene VPA tended to p e r s i s t longer i n plasma compared to VPA. The slower d e c l i n e of (E)-2-ene VPA from plasma may be due to the high degree of plasma p r o t e i n binding by t h i s compound. Plasma p r o t e i n binding of (E)-2-ene VPA i n r a t s i s concentration-dependent and v a r i e s between 90 ->99% at plasma concentrations ranging from 10 - 120 ug/mL (Semmes and Shen, 1990). An increase i n f r e e f r a c t i o n from 0.3% to 6.7% was observed as plasma (E)-2-ene VPA concentration increased from 10 to 60 ug/mL. A s i m i l a r trend was observed i n mice. As the plasma concentration of (E)-2-ene VPA increased from 30 to 55 ug/mL, plasma p r o t e i n binding was found to decrease from >98% to >93%, r e s p e c t i v e l y (Nau and Loscher, 1985). A s i m i l a r r a t i o n a l e can be used to e x p l a i n the r e s u l t s i n the current study of (E)-2-ene VPA pharmacokinetic p r o f i l e s i n r a t . Upon (E)-2-ene VPA a d m i n i s t r a t i o n , plasma concentrations reached a C m a x of approximately 140 ug/mL at which f r e e drug concentrations can be expected to be > 7%. The i n i t i a l phase of drug e l i m i n a t i o n appears to be r a p i d , but as drug plasma concentration decreases, the degree of p r o t e i n binding increases u n t i l i t reaches >99%. At that point with l e s s than 1% of f r e e (E)-2-ene VPA a v a i l a b l e i n plasma, the terminal e l i m i n a t i o n phase would be prolonged. Upon o r a l a d m i n i s t r a t i o n of 50 mg/kg of (E)-2-ene VPA to the mouse, a s i m i l a r d i s t r i b u t i o n phase as observed here i n the r a t , was reported but the prolonged terminal e l i m i n a t i o n phase was absent (Nau and Loscher, 1985). This observed d i f f e r e n c e between animal species was most l i k e l y a r e s u l t of d i s s i m i l a r i t i e s i n plasma p r o t e i n binding of (E)-2-ene VPA. Although no binding studies f o r (E)-2-ene VPA i n mouse were a v a i l a b l e , binding of VPA i n mouse was reported to be 11% (Loscher, 1978). By e x t r a p o l a t i o n , one can estimate that plasma p r o t e i n binding of (E)-2-ene VPA i n the mouse would be around 50% or l e s s . Thus, a greater f r e e f r a c t i o n i n mouse t r a n s l a t e s i n t o more a v a i l a b l e drug i n plasma f o r d i s t r i b u t i o n or e l i m i n a t i o n as compared to that i n the r a t . The p e r s i s t e n c e of (E)-2-ene VPA observed i n r a t plasma i s i n keeping with the idea of t h i s unsaturated compound being the a c t i v e metabolite r e s p o n s i b l e f o r the " c a r r y - o v e r " e f f e c t of VPA. In a d d i t i o n , the slow b u i l d - u p of (E)-2-ene VPA i n plasma f o l l o w i n g VPA a d m i n i s t r a t i o n could a l s o e x p l a i n the delay i n anticonvulsant a c t i v i t y observed f o l l o w i n g an i n i t i a l dose of VPA. One study has attempted to t e s t t h i s hypothesis. In a recent study i n v o l v i n g amygdala-kindled r a t s administered 200 mg/kg of (E)-2-ene VPA i . p . , no c o r r e l a t i o n between plasma concentration and anticonvulsant a c t i v i t y was found (Loscher et al., 1988). Therefore, i t appears that the prolonged anticonvulsant e f f e c t of (E)-2-ene VPA was not a manifestation of drug p e r s i s t i n g i n plasma. Perhaps a c o r r e l a t i o n between drug l e v e l and pharmacological e f f e c t e x i s t s i n other t i s s u e s such as the b r a i n . 4 . 4 . 2 P r o f i l e of (E)-2-ene VPA i n Peripheral Tissues No i n d i c a t i o n s were found to suggest that (E)-2-ene VPA p e r s i s t s or binds to peripheral t i s s u e s . The tissue/plasma r a t i o s i n l i v e r , kidneys, h e a r t , and lungs were found to decrease over the time of t m a x to tjQh ( f i g u r e 61). While the k i n e t i c p r o f i l e s of (E)-2-ene VPA i n l i v e r , kidneys, h e a r t , l u n g s , and plasma were s i m i l a r , the concentrations of drug i n the t i s s u e s were c o n s i s t e n t l y l e s s than that of plasma ( f i g u r e 24). There was a b r i e f increase i n (E)-2-ene VPA concentration i n l i v e r at 30 minutes that was most l i k e l y due to changes i n plasma f r e e drug c o n c e n t r a t i o n s . At higher drug concentrations the f r e e f r a c t i o n w i l l increase thereby a l l o w i n g more drug a v a i l a b l e f o r d i s t r i b u t i o n . As the f r e e f r a c t i o n of (E)-2-ene VPA decreases i n plasma the concentration i n l i v e r w i l l also decrease. A comparison of the l i v e r / p l a s m a r a t i o s at t m a x and tjoh showed a decrease from 1.4 to 0 . 5 , r e s p e c t i v e l y . S i m i l a r changes i n the tissue/plasma r a t i o s f o r kidneys, l u n g s , and heart were a l s o noted. The r e s u l t s of t h i s study of (E)-2-ene VPA were comparable to p r e v i o u s l y reported work i n which the d i s t r i b u t i o n k i n e t i c s of (E)-2-ene VPA were determined i n mouse (Nau and Loscher, 1985). The concentration of (E)-2-ene VPA i n mouse l i v e r was found to be l e s s than that i n plasma and the d e c l i n e of (E)-2-ene VPA from l i v e r p a r a l l e l e d the d e c l i n e from plasma. Based on these r e s u l t s i t was suggested that perhaps (E)-2-ene VPA could be used as an a l t e r n a t e anticonvulsant agent without the h e p a t o t o x i c i t y r e a c t i o n s associated with VPA (Nau and Loscher, 1985). Our r e s u l t s , from the metabolism of (E)-2-ene VPA that w i l l be discussed l a t e r , show that t h i s unsaturated metabolite may not be completely devoid of VPA-induced h e p a t o t o x i c i t y . 4 . 4 . 3 P r o f i l e of (E)-2-ene VPA i n Brain The uptake of (E)-2-ene VPA i n t o the whole b r a i n was found to reach a maximum concentration of 68.8 ± 19.8 ug/g w i t h i n 30 minutes. The p r o f i l e of d e c l i n e of (E)-2-ene VPA from whole b r a i n was s i m i l a r to that i n plasma and peripheral t i s s u e s . Concentrations of (E)-2-ene VPA i n b r a i n were lower than plasma with the AUCg-ioh values i n whole brain being one-tenth the values obtained f o r plasma. Brain/plasma r a t i o s f o r (E)-2-ene VPA i n whole b r a i n at t m a x and tjoh w e r e found to decrease from 0 . 3 to 0.04 r e s p e c t i v e l y and hence, s e l e c t i v e binding to b r a i n t i s s u e was not observed. These brain/plasma r a t i o s f o r (E)-2-ene VPA i n r a t were comparable to values of 0.02 - 0.09 obtained i n the dog 7 hours a f t e r i . v . a d m i n i s t r a t i o n of (E)-2-ene VPA (Loscher and Nau, 1983). The k i n e t i c p r o f i l e s and d i s t r i b u t i o n of (E)-2-ene VPA were a l s o determined i n various regions of the b r a i n . The regions analyzed were the same as those monitored f o l l o w i n g VPA a d m i n i s t r a t i o n . The k i n e t i c p r o f i l e s of (E)-2-ene VPA i n each b r a i n s e c t i o n were s i m i l a r as were the AUCo-ioh v a l u e s . Peak (E)-2-ene VPA l e v e l s i n the b r a i n regions were reached w i t h i n 30 minutes with concentrations i n the range of 30.0 -52.8 ug/g ( f i g u r e 6 2 ) . S e l e c t i v e t i s s u e binding was not observed as brain/plasma r a t i o s f o r (E)-2-ene VPA i n the various b r a i n regions at ^max a n d tjoh w e r e i n the range of 0 . 3 - 0.4 and 0.02 - 0.03 r e s p e c t i v e l y , s i m i l a r to those found i n whole brain ( f i g u r e 6 3 ) . D i s t r i b u t i o n of (E)-2-ene VPA i n the various regions of the brain appeared uniform, and none of the b r a i n s e c t i o n s were seen to accumulate t h i s drug. The p e r s i s t e n c e of (E)-2-ene VPA i n brain was a t t r i b u t e d to the prolonged and elevated mean plasma concentration of the drug observed at 10 hours. Even though (E)-2-ene VPA, at the lower c o n c e n t r a t i o n s , i s e l i m i n a t e d more slowly than VPA from b r a i n , the concentration of (E)-2-ene VPA observed at 10 hours was not c o n s i s t e n t with e f f e c t i v e anticonvulsant a c t i v i t y . Therefore, based on t h i s s i n g l e dose study i n r a t , i t i s h i g h l y u n l i k e l y that (E)-2-ene VPA i s the metabolite r e s p o n s i b l e f o r the post-drug e f f e c t of VPA. 4.4.4 Evaluating The Differences Between (E)-2-ene VPA and VPA i n Rats Because the plasma p r o t e i n binding of (E)-2-ene VPA i s s i g n i f i c a n t l y greater than that of VPA, the k i n e t i c p r o f i l e s of the compounds i n plasma can be expected to be d i s s i m i l a r . This d i f f e r e n c e can be seen during the terminal e l i m i n a t i o n phase, 240 minutes a f t e r drug a d m i n i s t r a t i o n , when the concentration of (E)-2-ene VPA i n plasma was greater than that of VPA ( f i g u r e 28). In peripheral t i s s u e s most notably i n l i v e r , the concentration of (E)-2-ene VPA was lower than that of VPA f o l l o w i n g equivalent doses. The l i v e r / p l a s m a r a t i o s f o r (E)-2-ene VPA at tjoh w e r e found to be s i g n i f i c a n t l y l e s s than VPA - a p o s s i b l e i n d i c a t i o n that (E)-2-ene VPA w i l l be l e s s hepatotoxic than VPA. In b r a i n , the mean concentrations of VPA and (E)-2-ene VPA at t m a x were s i m i l a r ( t a b l e 11). A f t e r 10 hours the concentrations of (E)-2-ene VPA were twice those of VPA throughout the various regions of the b r a i n ( f i g u r e 6 2 ) . The prolonged higher l e v e l s of (E)-2-ene VPA i n b r a i n were merely a r e f l e c t i o n of plasma concentrations and not s e l e c t i v e t i s s u e b i n d i n g . The brain/plasma r a t i o s at 10 hours between VPA and (E)-2-ene VPA were comparable ( f i g u r e 6 3 ) . 4 . 5 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E,E)-2,3'-DIENE VPA IN RATS 4 . 5 . 1 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Plasma In humans ( E , E ) - 2 , 3 ' - d i e n e VPA was found to be a major metabolite of VPA with serum concentrations between 6.4 - 7.1% that of VPA (Abbott et al., 1986; Kassahun et al., 1990). In r a t s 2 , 3 ' - d i e n e VPA was reported to be a major metabolite i n urine and plasma f o l l o w i n g VPA a d m i n i s t r a t i o n although the stereochemistry of the diene was not determined (Granneman et al., 1984; Loscher et al., 1988). An isomeric mixture c o n s i s t i n g of mainly (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n a 2:1 r a t i o was found to possess anticonvulsant a c t i v i t y i n mouse (Abbott and Acheampong, 1988). It was therefore proposed that the unusual pharmacodynamic a c t i v i t y of VPA may be r e l a t e d to the metabolite (E ,E)-2 , 3 ' - d i e n e VPA. If the metabolism i s s u f f i c i e n t l y d i f f e r e n t the diene may a l s o prove to be an a l t e r n a t i v e anticonvulsant drug to VPA. Following an i . p . dose of 150 mg/kg of ( E , E ) - 2 , 3 ' - d i e n e VPA peak plasma concentrations of 168.2 ± 13.9 ug/mL were a t t a i n e d w i t h i n 15 minutes. The plasma l e v e l of ( E , E ) - 2 , 3 ' - d i e n e VPA remained elevated f o r approximately 60 minutes which was then followed by a r a p i d d e c l i n e ( f i g u r e 26) . Unlike VPA or (E)-2-ene VPA, a secondary increase i n (E ,E)-2 , 3 ' - d i e n e VPA plasma concentration d i d not o c c u r , although there was a f l a t t e n i n g of the e l i m i n a t i o n curve at approximately 240 minutes post-dose. This was a t t r i b u t e d to the f a c t that the enteroheptic r e c y c l i n g of ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s was not as extensive as VPA or (E)-2-ene VPA (Lee, 1987). A h a l f - l i f e value f o r ( E , E ) - 2 , 3 ' - d i e n e VPA could not be determined because of the n o n - l i n e a r nature of the d a t a . In order to estimate the pharmacokinetic parameters from time zero to i n f i n i t y a h a l f - l i f e value of 28 minutes f o r the diene from a previous study i n r a t s was used (Lee, 1987). The AUC, C l / F , and Vj values from time zero to i n f i n i t y were c a l c u l a t e d to be 401 ug*h/mL, 6 mL/h/kg, and 378 mL/kg, r e s p e c t i v e l y . These values were comparable to those obtained i n the previous study (Lee, 1987). Values of 2.6 - 5.3 mL/h/kg and 172.5 - 279.4 mL/kg were reported f o r Cl and V j , r e s p e c t i v e l y . Plasma data f o r ( E , E ) - 2 , 3 ' - d i e n e VPA should p r e d i c t the t i s s u e l e v e l s of t h i s diene s i m i l a r to that observed f o r VPA and (E)-2-ene VPA. Thus, the r e l a t i v e l y r a p i d d e c l i n e of ( E , E ) - 2 , 3 ' - d i e n e VPA compared to VPA or (E)-2-ene VPA i n d i c a t e d that t h i s diene was also e l i m i n a t e d promptly from t i s s u e s . 4 . 5 . 2 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Peripheral Tissues Tissue d i s t r i b u t i o n of the a c t i v e VPA m e t a b o l i t e , ( E , E ) - 2 , 3 ' - d i e n e VPA, i n man and animals has not p r e v i o u s l y been determined. Because ( E , E ) - 2 , 3 ' - d i e n e VPA has p o t e n t i a l a p p l i c a t i o n as an anticonvulsant drug, the pharmacokinetic p r o f i l e s of t h i s agent i n the major t i s s u e s of the r a t were determined. The k i n e t i c p r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t l i v e r , kidneys, h e a r t , l u n g s , and plasma were s i m i l a r ( f i g u r e 26). D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA to the t i s s u e s from plasma was not r a p i d as the tmax values ranged from 15 - 60 minutes. The d e c l i n e phase of (E ,E)-2 , 3 ' - d i e n e VPA i n peripheral t i s s u e s appeared to p a r a l l e l the r a p i d d e c l i n e of the diene from plasma. The r a t e of d e c l i n e of the terminal e l i m i n a t i o n phase f o r ( E , E ) - 2 , 3 ' - d i e n e VPA i n peripheral t i s s u e s was f a s t e r than e i t h e r that of VPA or (E)-2-ene VPA ( f i g u r e s 29 - 3 2 ) . Because plasma p r o t e i n binding of ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t was found to be <90% at plasma concentrations of approximately 60 ug/mL (table 13), the increased a v a i l a b i l i t y of f r e e diene would account f o r i t s f a s t e r d e c l i n e i n plasma and u l t i m a t e l y i n peripheral t i s s u e s . A secondary increase i n t i s s u e diene concentrations was noted i n lungs and to some extent i n kidneys ( f i g u r e 2 6 ) , even though evidence f o r enterohepatic r e c y c l i n g i n plasma was not very s i g n i f i c a n t , u n l i k e that of VPA or (E)-2-ene VPA. Thus, the b r i e f increase i n ( E , E ) - 2 , 3 ' -diene VPA concentration i n lungs during the terminal e l i m i n a t i o n phase was unusual and the mechanism i s u n c e r t a i n . The r e l a t i v e l y r a p i d d e c l i n e of ( E , E ) - 2 , 3 ' - d i e n e VPA from l i v e r , compared to VPA or (E)-2-ene VPA ( f i g u r e 29) , together with the small t issue/plasma r a t i o c a l c u l a t e d at the 10 hour time point f o l l o w i n g drug a d m i n i s t r a t i o n suggests t h i s diene may be p o t e n t i a l l y l e s s hepatotoxic than VPA. Further evidence i n support of t h i s hypothesis w i l l r e q u i r e an examination of the metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s with the aim of i d e n t i f y i n g p o s s i b l e r e a c t i v e products. 4.5.3 P r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n Brain There are p r e s e n t l y no studies that describe the k i n e t i c s or d i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA i n brain f o r purposes of e s t a b l i s h i n g whether the diene favors or p e r s i s t s i n d i s c r e t e areas of the b r a i n . Only one report i n the l i t e r a t u r e examined the presence of 2 , 3 ' - d i e n e VPA i n the s u b s t a n t i a n i g r a of r a t s f o l l o w i n g VPA a d m i n i s t r a t i o n (Loscher et al., 1988). Therefore, the k i n e t i c p r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n b r a i n was i n v e s t i g a t e d with the aim of whether the diene could be associated with the unusual pharmacodynamic e f f e c t s of VPA. No evidence was obtained to i n d i c a t e any such involvement. From the k i n e t i c p r o f i l e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n whole b r a i n , e l i m i n a t i o n appeared to be r a p i d compared to that of VPA or (E)-2-ene VPA. Based on the A U C o - i o h values the amount of ( E , E ) - 2 , 3 ' - d i e n e VPA present i n whole b r a i n was one-tenth that of plasma. Brain/plasma r a t i o s were g e n e r a l l y low ( f i g u r e 61) and were found to decrease with t i m e . In f a c t , the r a t i o s obtained f o r the diene i n brain were s i m i l a r to that of heart ( f i g u r e 61). No evidence was obtained to i n d i c a t e that ( E , E ) - 2 , 3 ' -diene VPA was bound to whole brain t i s s u e . S i m i l a r k i n e t i c p r o f i l e s were observed i n the various s e c t i o n s of b r a i n that were analyzed ( f i g u r e 27). D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' - d i e n e VPA throughout the b r a i n appeared uniform as the C m a x values obtained from the d i f f e r e n t regions were comparable to the value f o r whole b r a i n ( f i g u r e 62) . What was d i f f e r e n t was an increase i n the brain/plasma r a t i o s at 10 hours i n a l l regions examined except the putamen with the l a r g e s t increases observed i n the superior c o l l i c u l u s ( 0 . 6 ) , i n f e r i o r c o l l i c u l u s ( 1 . 4 ) , and s u b s t a n t i a n i g r a ( 0 . 8 ) ( f i g u r e 6 3 ) . Also noted i n the b r a i n s e c t i o n s was a t r a n s i e n t increase i n ( E , E ) - 2 , 3 ' - d i e n e VPA t i s s u e concentration 60 minutes f o l l o w i n g drug a d m i n i s t r a t i o n . Although unexplained, the increase was not l i k e l y an a n a l y t i c a l a r t i f a c t since t h i s d i d not occur i n other t i s s u e s or plasma assayed. A s i m i l a r r e s u l t was reported f o r repeated i . p . a d m i n i s t r a t i o n of VPA to r a t s f o r 3 days. The concentration of 2 , 3 ' - d i e n e VPA, thought to be the ( E , E ) - i s o m e r , i n the s u b s t a n t i a n i g r a was found to increase on the t h i r d day of VPA a d m i n i s t r a t i o n (Loscher et a 7 . , 1988). Increased e l e c t r i c a l a c t i v i t y i n the s u b s t a n t i a n i g r a appears to be a common feature of g e n e r a l i z e d seizures (Gale, 1988). VPA has been shown to i n h i b i t excessive neuronal a c t i v i t y (Waczcak et al., 1986) and increase GABA concentration i n the nerve terminals w i t h i n the s u b s t a n t i a n i g r a of r a t s (Iadarola and Gale, 1981; Loscher and V e t t e r , 1984). The present study has shown that f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n to r a t s , t h i s compound binds s e l e c t i v e l y to c e r t a i n regions of the b r a i n . Tissue-to-plasma r a t i o s i n these areas were found to increase over t i m e . Thus, the p e r s i s t e n c e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n b r a i n may i n part e x p l a i n the prolonged duration of a c t i v i t y observed f o l l o w i n g the d i s c o n t i n u a t i o n of VPA. However, one must consider that brain l e v e l s of the diene decreased r a p i d l y over the 10 hour period f o l l o w i n g the dose and concentrations i n the brain s e c t i o n s at 10 hours were very low (<0.1 ug/g). 4 .5.4 Evaluating D i f f e r e n c e s i n the Tissue D i s t r i b u t i o n of ( E , E ) - 2 , 3 ' -diene VPA t o VPA and (E)-2-ene VPA Following s i n g l e dose ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , the d e c l i n e of drug from plasma during the terminal e l i m i n a t i o n phase was f a s t e r than that of VPA. The c e n t r a l compartment f o r t h i s diene appears to i n c l u d e t i s s u e s as well as plasma and hence, the e l i m i n a t i o n p r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA from these t i s s u e s p a r a l l e l s that i n plasma. Based on tissue/plasma r a t i o s , the binding of ( E , E ) - 2 , 3 ' - d i e n e VPA to l i v e r t i s s u e s at the 10 hour time point was s i g n i f i c a n t l y l e s s than that of VPA ( f i g u r e 61). Since the p e r s i s t e n c e or binding of VPA to l i v e r may be a prelude to an i d i o s y n c r a t i c hepatotoxic r e a c t i o n , the t r a n s i e n t nature of t h i s diene i n l i v e r could avoid s i m i l a r c o m p l i c a t i o n s . U n f o r t u n a t e l y , ( E , E ) - 2 , 3 ' - d i e n e VPA was seen to d e c l i n e r a p i d l y from whole b r a i n and hence, the duration of pharmacological a c t i v i t y would be l i m i t e d . There were some i n d i c a t i o n s of s i t e s p e c i f i c binding of (E ,E)-2 , 3 ' - d i e n e VPA to the s u p e r i o r c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , and s u b s t a n t i a n i g r a which could be i n t e r p r e t e d as p o t e n t i a l areas of drug a c t i o n . However, the absolute concentration of t h i s diene i n these areas appeared too low to exert any pharmacological e f f e c t . D i f f e r e n c e s i n the k i n e t i c p r o f i l e s between ( E , E ) - 2 , 3 ' - d i e n e VPA and (E)-2-ene VPA i n plasma were s i m i l a r to those found between (E ,E)-2 , 3 ' - d i e n e VPA and VPA. A s t r i k i n g d i f f e r e n c e i s the terminal d e c l i n e phase of ( E , E ) - 2 , 3 ' - d i e n e VPA from plasma being f a s t e r than e i t h e r VPA or (E)-2-ene VPA. The plasma p r o t e i n binding p r o p e r t i e s of these compounds may l a r g e l y be responsible f o r these d i f f e r e n c e s . 4 . 6 TISSUE DISTRIBUTION AND KINETIC PROFILES OF VPA METABOLITES IN RATS 4 . 6 . 1 P r o f i l e s of VPA Metabolites i n Plasma The metabolism of VPA has been e x t e n s i v e l y studied (Ferrandes and Eymard, 1977; Acheampong et al., 1983; Turnbull et al., 1983; Granneman et al., 1984; Heinemeyer et al., 1985; Rettenmeier et al., 1987; Rogiers et al., 1988). Several pathways are involved i n the metabolism of VPA ( f i g u r e s 3 and 4) although glucuronidation and 16-oxidation are considered the main routes (Granneman et al., 1984). A major metabolite present i n humans and animals that i s not a d i r e c t product of e i t h e r of the two major pathways i s 2 , 3 ' - d i e n e VPA (Abbott et al., 1986). An e a r l i e r study i n v o l v i n g the i d e n t i f i c a t i o n of VPA metabolites i n p a t i e n t s by GC-MS described the i s o l a t i o n of two diene metabolites postulated to have e i t h e r the ct,fi'- or fi,y'-configuration (Acheampong et al., 1983). In a p a t i e n t who had succumbed to VPA h e p a t o t o x i c i t y , f i v e diunsaturated metabolites of VPA were observed i n plasma and urine with two i d e n t i f i e d as 4 , 4 ' - d i e n e VPA and (E)-2 ,4-diene VPA (Kochen et al., 1984). The remaining three compounds were l a b e l l e d as d i e n e s / 2 , / 3 , and / 4 . I t was not u n t i l authentic samples of these unknown compounds were synthesized that the stereochemical and p o s i t i o n a l isomers were determined to be (E)- and ( Z ) - 2 , 4 - d i e n e VPA and, (E ,E)- and ( E , Z ) - 2 , 3 ' -diene VPA (Acheampong and Abbott, 1985; Lee et al., 1989). 180 The metabolites i d e n t i f i e d f o l l o w i n g i . p . a d m i n i s t r a t i o n of 150 mg/kg of VPA to r a t s were s i m i l a r to those reported by Loscher et al. (1988). In Loscher's i n v e s t i g a t i o n s the only diene detected i n plasma was reported to be a 2 , 3 ' - d i e n e VPA with the stereochemistry not determined. In our study by using an improved method f o r the a n a l y s i s of VPA metabolites and having s y n t h e t i c standards a v a i l a b l e , three diene metabolites were i d e n t i f i e d . The metabolites detected i n plasma were ( E , E ) - 2 , 3 ' - d i e n e VPA, ( E , Z ) - 2 , 3 ' - d i e n e VPA, and ( E ) - 2 , 4 - d i e n e VPA ( f i g u r e 21). The ( E , Z ) - 2 , 3 ' - d i e n e VPA was present i n r a t plasma at low concentrations ( f i g u r e 34) . This metabolite had not been p r e v i o u s l y i d e n t i f i e d i n the r a t and information regarding the anticonvulsant a c t i v i t y , t o x i c i t y , or pharmacokinetic c h a r a c t e r i s t i c s of t h i s diene were not known. Unusually high amounts of 3-ene VPA i n plasma comparable to that of (E)-2-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA were noted. Rats appear to produce a greater amount of 3-ene VPA from VPA compared to man. The 3 -ene VPA i s g e n e r a l l y a minor metabolite of VPA i n humans (Abbott et al., 1986; Kassahun et al., 1990). The o r i g i n of 3-ene VPA i s p r e s e n t l y unknown although i t has been suggested that 3-ene VPA i s a d i r e c t metabolite of (E)-2-ene VPA via an isomerase enzyme (Rettenmeier et al., 1987). The anticonvulsant a c t i v i t y of 3-ene VPA i n the mouse was found to be 50% that of (E)-2-ene VPA and hence, the c o n t r i b u t i o n of 3-ene VPA to VPA anticonvulsant a c t i v i t y would probably be i n s i g n i f i c a n t (Loscher and Nau, 1985). 181 4 . 6 . 2 P r o f i l e of VPA Metabolites i n Peripheral Tissues The metabolites observed i n l i v e r , kidneys, h e a r t , and lungs f o l l o w i n g VPA a d m i n i s t r a t i o n were very s i m i l a r to those found i n plasma. Of p a r t i c u l a r i n t e r e s t was the l i v e r since VPA produces an i d i o s y n c r a t i c hepatotoxic r e a c t i o n c h a r a c t e r i z e d by m i c r o v e s i c u l a r s t e a t o s i s and n e c r o s i s (Zimmerman and Ishak, 1982). The unsaturated metabolites (E)-2 , 4 - d i e n e VPA and 4-ene VPA were shown to induce a s i m i l a r pathology when administered to r a t s (Kesterson et al., 1984). A f t e r VPA, very low concentrations of 4-ene VPA were seen i n the l i v e r ( f i g u r e 3 6 ) . In s p i t e of ( E ) - 2 , 4 - d i e n e VPA exceeding the concentration of 4-ene VPA i n plasma, kidneys, and lungs ( f i g u r e 34, appendices 10 and 12), no evidence of t h i s diene could be found i n the l i v e r . The i n a b i l i t y to detect ( E ) - 2 , 4 - d i e n e VPA from l i v e r may be due to i t s covalent binding to endogenous l i v e r components. Evidence obtained by Kassahun (1991) f o l l o w i n g the a d m i n i s t r a t i o n of ( E ) - 2 , 4 - d i e n e VPA to r a t s suggests that the d e t o x i f i c a t i o n mechanism of the l i v e r may be producing a h i g h l y r e a c t i v e e l e c t r o p h i l i c intermediate p o t e n t i a l l y capable of i r r e v e r s i b l y a l k y l a t i n g key l i v e r enzymes. While not s p e c i f i c a l l y looked f o r i n the current study, the g l u t a t h i o n e conjugate of ( E ) - 2 , 4 - d i e n e VPA has been detected i n r a t b i l e and the N-a c e t y l c y s t e i n e conjugate i d e n t i f i e d i n the urine of p a t i e n t s on VPA. This i d e n t i f i c a t i o n of these conjugates i s strong evidence i n favor of the formation of a r e a c t i v e e l e c t r o p h i l i c s p e c i e s . Thus, the absence of ( E ) - 2 , 4 - d i e n e VPA i n l i v e r t i s s u e may be an i n d i c a t i o n of i r r e v e r s i b l e t i s s u e b i n d i n g . 4 . 6 . 3 P r o f i l e of VPA Metabolites i n Brain E a r l i e r studies had shown that f o l l o w i n g chronic o r a l a d m i n i s t r a t i o n of VPA to mice the only metabolite observed i n whole b r a i n was (E)-2-ene VPA. Two days f o l l o w i n g the d i s c o n t i n u a t i o n of VPA, (E)-2-ene VPA could s t i l l be detected i n b r a i n a l b e i t at low l e v e l s (Loscher and Nau, 1982). A s i m i l a r study i n dogs i n which VPA was administered as an i . v . bolus and i n r a t s where VPA was dosed v i a an osmotic mini-pump, (E)-2-ene VPA was a l s o found i n the b r a i n (Loscher and Nau, 1983). More s p e c i f i c a l l y , i n r a t b r a i n (E)-2-ene VPA was most prominent i n hippocampus, superior and i n f e r i o r c o l l i c u l u s , s u b s t a n t i a n i g r a , and medulla. It has been speculated that the s u b s t a n t i a n i g r a may be the s i t e of anticonvulsant a c t i o n since t h i s area i s associated with high GABA a c t i v i t y ( I a d a r o l a and Gale, 1982). The accumulation of a c t i v e VPA metabolites f o l l o w i n g VPA a d m i n i s t r a t i o n i n the s u b s t a n t i a n i g r a would support the above hypothesis. In a recent study i n r a t s where m u l t i p l e doses of 200 mg/kg of VPA was administered i . p . , no accumulation of (E)-2-ene VPA i n b r a i n , i n c l u d i n g the s u b s t a n t i a n i g r a , was observed (Loscher et al., 1988). This was i n contrast to an e a r l i e r study i n r a t s administered VPA by osmotic mini pumps. The d i f f e r e n c e s i n r e s u l t s were probably due to the low plasma l e v e l s of VPA achieved, 14 - 20 ug/mL, using the mini pump versus the i . p . a d m i n i s t r a t i o n (Loscher and Nau, 1983). As d e t e c t i o n methods f o r VPA metabolites improved and plasma concentrations of VPA were i n c r e a s e d , a greater number of metabolites was observed i n the b r a i n . In r a t s administered m u l t i p l e doses of VPA, s i x metabolites could be detected i n the s u b s t a n t i a n i g r a (Loscher et al., 1988). These metabolites were, i n order of decreasing c o n c e n t r a t i o n , 5-OH VPA, 3-keto VPA, 4-OH VPA, 3-ene VPA, 2 , 3 ' - d i e n e VPA (stereochemistry not known), and (E)-2-ene VPA. In the current study comparable r e s u l t s were obtained. Because the s e n s i t i v i t y and s t e r e o s e l e c t i v i t y of our assay was s i g n i f i c a n t l y improved, several more metabolites were observed i n b r a i n . These were 4-keto VPA, 3-OH VPA, 2-PGA, and 4-ene VPA. No evidence of VPA metabolites p e r s i s t i n g i n the s u b s t a n t i a n i g r a over the 10 hour period was observed. The d e c l i n e of the metabolites from the s u b s t a n t i a n i g r a was s i m i l a r to that of the parent compound ( f i g u r e s 38 and 3 9 ) . Q u a n t i t a t i o n of VPA metabolites i n other regions of the b r a i n , such as the hippocampus, s u p e r i o r c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , cerebellum, o l f a c t o r y b u l b s , corpus c a l l o s u m , medulla, and putamen, gave r e s u l t s i d e n t i c a l to that of the s u b s t a n t i a n i g r a (appendices 17 - 32) . The k i n e t i c p r o f i l e s of VPA metabolites i n b r a i n were s i m i l a r to those i n plasma, although ( E ) - 2 , 4 - d i e n e VPA was absent i n b r a i n . Most s u r p r i s i n g was the d e t e c t i o n i n brain of ( E , Z ) - 2 , 3 ' - d i e n e VPA, a minor diene metabolite i n plasma. Also s u r p r i s i n g was the complete absence of ( E , E ) - 2 , 3 ' - d i e n e VPA, the major metabolite of VPA i n plasma. In an attempt to e x p l a i n t h i s unusual f i n d i n g the f o l l o w i n g p o s s i b i l i t i e s were considered. The (E,Z)- and (E,E)-isomers of the diene appear to i l l u s t r a t e d i s t i n c t stereochemical p r o p e r t i e s . Perhaps (E ,Z)-2 , 3 ' - d i e n e VPA binds favorably to brain t i s s u e while the (E ,E)-isomer has a low a f f i n i t y or f a i l s even to cross the blood brain b a r r i e r . However, upon subsequent a d m i n i s t r a t i o n of the ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s , t h i s diene was r e a d i l y detected i n b r a i n and could s t i l l be detected 10 hours f o l l o w i n g the dose. Another p o s s i b i l i t y f o r the presence of ( E , Z ) - 2 , 3 ' - d i e n e VPA and not the (E ,E)-isomer i n the s u b s t a n t i a n i g r a could be due to d i f f e r e n c e s i n plasma p r o t e i n b i n d i n g . The ( E , Z ) - 2 , 3 ' - d i e n e VPA was 28.7 - 75.8% bound to plasma p r o t e i n s compared to 74.2 - 97.1% f o r the (E ,E)-isomer ( t a b l e 13). The amount of f r e e ( E , Z ) - 2 , 3 ' - d i e n e VPA a v a i l a b l e i n plasma f o r d i s t r i b u t i o n i n t o b r a i n would be greater than that of ( E , E ) - 2 , 3 ' -diene VPA. However, i f p r o t e i n binding was the major f a c t o r r e s p o n s i b l e f o r the absence of ( E , E ) - 2 , 3 ' - d i e n e VPA i n b r a i n , t h i s phenomenon should a l s o occur i n the other t i s s u e s assayed. Furthermore, since (E)-2-ene VPA i s h i g h l y plasma p r o t e i n bound (Semmes and Shen, 1990) t h i s metabolite should a l s o be absent from brain t i s s u e s . This was not the c a s e . Thus, the absence of ( E , E ) - 2 , 3 ' - d i e n e VPA i n b r a i n cannot be e a s i l y r a t i o n a l i z e d based on plasma p r o t e i n b i n d i n g . A t h i r d p o s s i b i l i t y was that ( E , E ) - 2 , 3 ' - d i e n e VPA was isomerizing to the (E ,Z)-isomer i n b r a i n . The l i k e l i h o o d of t h i s having occurred was s m a l l . Following the a d m i n i s t r a t i o n of (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n a 19:1 r a t i o r e s p e c t i v e l y to r a t s , s i m i l a r r a t i o s of the dienes were observed throughout the b r a i n . A l i k e l y explanation f o r the absence of ( E , E ) - 2 , 3 ' - d i e n e VPA from b r a i n f o l l o w i n g VPA a d m i n i s t r a t i o n , i s the presence of a s e l e c t i v e t r a n s p o r t mechanism r e s p o n s i b l e f o r r a p i d l y removing the diene out of the b r a i n , s i m i l a r to that proposed f o r VPA (Cornford et a 7 . , 1985). This would account f o r the r a p i d removal of ( E , E ) - 2 , 3 ' - d i e n e VPA from b r a i n f o l l o w i n g a d m i n i s t r a t i o n of the diene i t s e l f . Upon ( E , E ) - 2 , 3 ' -diene VPA a d m i n i s t r a t i o n , the i n i t i a l l y high diene plasma concentrations would most l i k e l y saturate t h i s t r a n s p o r t mechanism thereby a l l o w i n g f o r some of the compound to i n i t i a l l y remain i n b r a i n . The d i f f e r e n c e s i n d e c l i n e f o r (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA from whole b r a i n f o l l o w i n g the simultaneous a d m i n i s t r a t i o n of both compounds i n a 19:1 r a t i o , r e s p e c t i v e l y i s supportive of t h i s hypothesis ( f i g u r e 5 0 ) . The d e c l i n e of ( E , E ) - 2 , 3 ' - d i e n e VPA from whole brain was c l e a r l y f a s t e r than that of the (E,Z)-isomer with the d e c l i n e curves having crossed a f t e r 200 minutes f o l l o w i n g a d m i n i s t r a t i o n . On the other hand, the d e c l i n e p r o f i l e s ( p a r a l l e l curves) of both dienes appeared to be s i m i l a r i n the s u b s t a n t i a n i g r a ( f i g u r e 51) and i n a l l other brain regions (appendices 64 - 71). This could be expected because d e c l i n e of the dienes from i n d i v i d u a l b r a i n regions may not represent drug c l e a r i n g from the b r a i n i t s e l f , whereas whole b r a i n a n a l y s i s b e t t e r r e f l e c t s the 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 the isomers from the b r a i n . Studies examining the t r a n s p o r t of both dienes across the b l o o d - b r a i n b a r r i e r bears f u r t h e r i n v e s t i g a t i o n . 4.7 TISSUE DISTRIBUTION AND KINETIC PROFILES OF METABOLITES OF (E)-2-ENE VPA IN RATS 4 . 7 . 1 P r o f i l e s of (E)-2-ene VPA Metabolites i n Plasma The VPA m e t a b o l i t e , (E)-2-ene VPA i s c u r r e n t l y undergoing c l i n i c a l t r i a l s i n Europe with the aim of marketing t h i s compound as an a l t e r n a t e anticonvulsant drug to VPA. While h e p a t o t o x i c i t y (Dreifuss et a / . , 1989) and embryotoxicity ( B e r t o l l i n i et a 7 . , 1985; Kaneko et a 7 . , 1988; Carter and Stewart, 1989) are negative keynote features of VPA, (E)-2-ene VPA was found to have a s i g n i f i c a n t l y decreased t e r a t o g e n i c e f f e c t i n mice (Loscher et a 7 . , 1984; Loscher and Nau, 1985). Furthermore, f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n to mice, the concentration of t h i s compound i n l i v e r was s i g n i f i c a n t l y lower than that of VPA when given at comparable doses (Nau and Loscher, 1985). However, s i n c e 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA are the metabolites implicated i n VPA h e p a t o t o x i c i t y (Kassahun et a / . , 1989; Porubek et a l . , 1989) the degree of biotransformation of (E)-2-ene VPA to these t o x i c species may be a more appropriate i n d i c a t o r of p o t e n t i a l t o x i c i t y . For (E)-2-ene VPA to be considered l e s s hepatotoxic than VPA, v i r t u a l l y l i t t l e or no 4-ene VPA or (E)-2 ,4-diene VPA should a r i s e from (E)-2-ene VPA metabolism. But i n f a c t f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n to r a t s both 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA were detected i n plasma ( f i g u r e 40) at concentrations s i m i l a r to that f o l l o w i n g VPA a d m i n i s t r a t i o n ( f i g u r e 3 4 ) . Thus i f ( E ) - 2 , 4 - d i e n e VPA i s the key f a c t o r i n VPA h e p a t o t o x i c i t y , one would p r e d i c t s i m i l a r problems f o r the (E)-2-ene VPA. A summary of the metabolism f o r (E)-2-ene VPA determined from the metabolites detected i n plasma f o l l o w i n g drug a d m i n i s t r a t i o n i s i l l u s t r a t e d i n f i g u r e 64. The main pathways f o r the metabolism of (E)-2-ene VPA, based on the presence of each m e t a b o l i t e , appear to be /3-o x i d a t i o n , r e d u c t i o n , dehydrogenation, and i s o m e r i z a t i o n . These pathways account f o r most of the metabolites detected i n plasma f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n . Since the 0 - o x i d a t i o n pathway i s a known major pathway f o r VPA metabolism (Abbott et a 7 . , 1986), and (E)-2-ene VPA i s the i n i t i a l metabolite of t h i s pathway, i t was not s u r p r i s i n g to see that (E)-2-ene VPA was being metabolized by t h i s r o u t e . The reduction of (E)-2-ene VPA and other unsaturated f a t t y acids mediated by reductases i n r a t s has p r e v i o u s l y been reported (Granneman 187 3 - O H VPA -4- ( E ) - 2 - e n e VPA 3 - k e t o VPA ( E ) - 2 . 4 - d i e n e VPA J 4 - e n e VPA VPA *+• +~ ( E / Z ) - 3 - e n e VPA ( E E / E Z ) - 2 . 3 ' - d f e n e VPA 4 - O H - V P A 5 - O H VPA 1 2 - P G A Figure 64: Proposed metabolic scheme f o r (E)-2-ene VPA i n r a t s (dotted l i n e represents a p o s s i b l e pathway). et al., 1986). The formation of 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA f o l l o w i n g (E)-2-ene VPA i s not c l e a r . Two p o s s i b l e routes of metabolism have been proposed. The dehydrogenation of (E)-2-ene VPA to ( E , E ) - 2 , 3 ' -diene VPA followed by reduction to 3-ene VPA (Abbott et al., 1986) or the i s o m e r i z a t i o n of (E)-2-ene VPA to 3-ene VPA followed by /J-oxidation to ( E , E ) - 2 , 3 ' - d i e n e VPA (Rettenmeier et al., 1987). Both routes appear to be p o s s i b l e as 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA were present i n s i m i l a r amounts i n plasma, f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n . In a d d i t i o n , the a d m i n i s t r a t i o n of ( E , E ) - 2 , 3 ' - d i e n e a l s o r e s u l t e d i n both 3-ene VPA and (E)-2-ene VPA. Polar metabolites such as 4-OH VPA, 5-OH VPA, and 2-PGA, were l i k e l y secondary metabolites of (E)-2-ene VPA derived d i r e c t l y from VPA. In other words the VPA produced via the reduction of (E)-2-ene VPA underwent a second phase of metabolism to y i e l d products normally observed f o l l o w i n g VPA b i o t r a n s f o r m a t i o n . Other products from VPA metabolism not detected f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n were most l i k e l y below the l i m i t s of the assay. 4 . 7 . 2 P r o f i l e s of (E)-2-ene VPA Metabolites i n Peripheral Tissues VPA was the major metabolite observed i n l i v e r f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n ( f i g u r e 42). The concentration of VPA i n l i v e r was above 10 ug/mL over the e n t i r e 10 hour p e r i o d , 1 0 - f o l d greater than that i n plasma ( f i g u r e 40) . The 4-ene VPA concentration i n l i v e r f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n was approximately 1000 times greater than that observed i n l i v e r f o l l o w i n g VPA a d m i n i s t r a t i o n ( f i g u r e 3 6 ) . This suggests that 4-ene VPA i s formed by a d i f f e r e n t route rather than via microsomal o x i d a t i o n of VPA. There i s l i t t l e reason to t h i n k 4-ene VPA should be higher i n l i v e r exposed to l e s s e r amounts of VPA unless (E)-2-ene VPA was s a t u r a t i n g ^ - o x i d a t i o n , thereby shunting VPA i n t o the 4-ene VPA pathway. Elevated 4-ene VPA l i v e r concentrations f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n may have t o x i c o l o g i c a l i m p l i c a t i o n s . No ( E ) - 2 , 4 - d i e n e VPA was detected i n l i v e r f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n s i m i l a r to that observed upon VPA a d m i n i s t r a t i o n . As p r e v i o u s l y d i s c u s s e d , t h i s does not i n f e r that ( E ) - 2 , 4 - d i e n e VPA was not present i n l i v e r as ( E ) - 2 , 4 - d i e n e VPA i s a known metabolite of 4-ene VPA (Rettenmeier et al., 1985) and appears here to be a d i r e c t microsomal metabolite of (E)-2-ene VPA. The diene metabolite may i n f a c t have been i r r e v e r s i b l y conjugated to l i v e r components or reacted with g l u t a t h i o n e and thus not amenable to d e t e c t i o n . The d e t e c t i o n of unusually high concentrations of 4-ene VPA i n l i v e r and the r e l a t i v e l y high plasma l e v e l s of both 4-ene VPA and (E)-2 , 4 - d i e n e VPA f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n suggests that (E)-2-ene VPA may not be e n t i r e l y free of h e p a t o t o x i c i t y . On the c o n t r a r y , (E)-2-ene VPA may pose a p o t e n t i a l l y greater hazard than that of VPA. 4 . 7 . 3 P r o f i l e s of (E)-2-ene VPA Metabolites i n Brain The metabolites detected i n whole brain and the various brain regions were s i m i l a r to those observed i n plasma. The k i n e t i c p r o f i l e s of these metabolites appear to be s i m i l a r to the parent compound i n d i c a t i n g that the concentration of the metabolites was dependent on (E)-2-ene VPA c o n c e n t r a t i o n . There was no evidence i n d i c a t i n g the p e r s i s t e n c e of metabolite(s) i n brain f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n as the concentration of each metabolite at 10 hours was l e s s than that of the parent compound. VPA was a l s o detected as a metabolite of (E)-2-ene VPA i n the b r a i n at l e v e l s s i m i l a r to that of 3-ene VPA. Perhaps the anticonvulsant a c t i v i t y associated with (E)-2-ene VPA (Abbott and Acheampong, 1988) was due to VPA. However, the concentration of VPA i n b r a i n t i s s u e was l e s s than 1 ug/mL. This appeared to be too low to be c l i n i c a l l y s i g n i f i c a n t s i n c e amygdala-kindled r a t s with VPA brain concentrations of 1 ug/mL d i d not d i s p l a y any anticonvulsant a c t i v i t y (Loscher et al., 1988). 4 . 8 . TISSUE DISTRIBUTION AND KINETIC PROFILES OF METABOLITES OF ( E , E ) -2,3'-DIENE VPA IN RATS 4 . 8 . 1 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Plasma The reduction of ( E , E ) - 2 , 3 ' - d i e n e VPA appears to be the main route of metabolism as the major metabolites observed i n plasma were (E)-2-ene VPA and 3-ene VPA ( f i g u r e 6 5 ) . Other metabolites i d e n t i f i e d i n s i m i l a r amounts were VPA and 3-keto VPA, products of secondary metabolism of (E)-2-ene VPA. The mean plasma concentration of VPA observed f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n was l e s s than that observed upon (E)-2-ene VPA a d m i n i s t r a t i o n . VPA was f u r t h e r metabolized to y i e l d the p o l a r compounds, 4-OH VPA and 5-OH VPA. Other biotransformation products of VPA, such as 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA, were not detected p o s s i b l y because the concentrations of the l o g i c a l p r e c u r s o r s , VPA and (E)-2-ene VPA i n plasma were i n i t i a l l y low. This would suggest a lower p o t e n t i a l f o r l i v e r t o x i c i t y with t h i s diene over e i t h e r VPA or (E)-2-ene VPA. 2 - e n e - 4 ' - 0 H VPA 2 .4 ,3 , - t r i ene V P A N t / 3 - k e t o - 3 ' - e n e V P A I1TIZ 3 - 0 H - 3 ' - e n e VPA 2 - e n e - 3 ' - 0 H V P A ( E . E ) - 2 . 3 ' - d i e n e V P A 3 , 3 - d i e n e VPA / ( E ) - 2 - e n e V P A / \ ( E / Z ) - 3 - e n e V P A 3 - O H V P A V P A / 3 - k e t o V P A 5 - O H V P A 4 - 0 H V P A Figure 65: Proposed metabolic scheme f o r ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s (dotted l i n e represents p o s s i b l e metabolites or pathways). A metabolite observed i n plasma f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n that was not p r e v i o u s l y detected was a diene of unknown c o n f i g u r a t i o n . GC-MS a n a l y s i s i n d i c a t e d that t h i s compound chromatographed p r i o r to the two other known d i e n e s . The r e t e n t i o n time f o r t h i s unknown diene was approximately 8.01 minutes. Although the unknown diene could be any number of p o s s i b l e d i e n o i c isomers, based on the reported GC c h a r a c t e r i s t i c s of diene isomers of VPA, t h i s compound i s l i k e l y to be ( Z , Z ) - 3 , 3 ' - d i e n e VPA (Acheampong' and Abbott, 1985). U n t i l a synthesized standard f o r ( Z , Z ) - 3 , 3 ' - d i e n e VPA i s a v a i l a b l e , p o s i t i v e i d e n t i f i c a t i o n by GC-MS and NMR was not p o s s i b l e . I d e n t i f i c a t i o n of p o s s i b l e metabolites p r e d i c t e d f o r ( E , E ) - 2 , 3 ' -diene VPA metabolism, as i l l u s t r a t e d i n f i g u r e 65 was attempted, but no GC-MS peaks were detected that corresponded to the appropriate base i o n s . Since s y n t h e t i c standards f o r most of these p o t e n t i a l metabolites were not a v a i l a b l e , p o s i t i v e i d e n t i f i c a t i o n would not have been p o s s i b l e . Only the 3-0H-3'-ene VPA metabolite was synthesized but , upon a n a l y s i s of plasma samples, no peak corresponding to t h i s metabolite was observed. 4 . 8 . 2 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n Peripheral Tissues The e l i m i n a t i o n of the unsaturated and p o l a r metabolites from l i v e r f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n was slower than that of the parent compound. The concentration of diene f e l l to a l e v e l l e s s than that of i t s metabolites w i t h i n 180 minutes. Although the main metabolites detected i n plasma ( f i g u r e s 46 and 47) were a l s o found i n l i v e r ( f i g u r e s 48 and 4 9 ) , the mean VPA concentration i n l i v e r was 100 times that i n plasma. However, t h i s was not unusual as s i m i l a r f i n d i n g s f o r VPA i n l i v e r were observed f o l l o w i n g VPA or (E)-2-ene VPA a d m i n i s t r a t i o n ( f i g u r e 42). What was i n t e r e s t i n g was the absence of 4-ene VPA and presumably ( E ) - 2 , 4 - d i e n e VPA i n l i v e r . ( E ) - 2 , 4 - d i e n e VPA or 4-ene VPA are not d i r e c t by-products of ( E , E ) - 2 , 3 ' - d i e n e VPA biotransformation l i k e that of VPA ( f i g u r e 3) or (E)-2-ene VPA ( f i g u r e 6 4 ) . The metabolites observed i n kidneys, h e a r t , and lungs f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n were s i m i l a r to the l i v e r although the amount of VPA present i n these t i s s u e s was l e s s than that of l i v e r . In summary, the metabolites of ( E , E ) - 2 , 3 ' - d i e n e VPA observed i n p e r i p h e r a l t i s s u e s d i d not d i f f e r from those observed i n plasma. The m a j o r i t y of the metabolites detected appeared to be s i m i l a r to those observed f o l l o w i n g VPA a d m i n i s t r a t i o n . Of p a r t i c u l a r i n t e r e s t was the absence of 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA i n l i v e r f o l l o w i n g (E ,E)-2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , suggesting that t h i s diene may be f r e e of the hepatotoxic e f f e c t s associated with VPA. 4 . 8 . 3 P r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA Metabolites i n B r a i n The metabolites detected i n whole brain and i n the various b r a i n regions f o l l o w i n g ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n were s i m i l a r to those observed i n plasma ( f i g u r e s 46 and 47). S l i g h t l y higher concentrations of VPA were observed i n the s u b s t a n t i a n i g r a ( f i g u r e 51) and other b r a i n regions (appendices 64 - 71) compared to that of plasma. The higher concentrations of VPA found i n b r a i n r e l a t i v e to plasma suggests that reduction of ( E , E ) - 2 , 3 ' - d i e n e VPA was o c c u r r i n g i n b r a i n , but s i m i l a r r e s u l t s were not observed f o l l o w i n g (E)-2-ene VPA administration. Another p o s s i b i l i t y was that VPA was binding to brain tissue although t h i s was not observed following VPA administration. VPA , 5-OH VPA, and 3-keto VPA concentrations in the substantia nigra were found to increase over the 10 hour period (figures 51 and 52). Similar results were observed in the other brain regions analyzed. In spite of the persistence of 5-0H VPA and 3-keto VPA in brain, these compounds are not considered to be potent anticonvulsants (Loscher, 1981). The question arises whether VPA and/or (E)-2-ene VPA, major metabolites in brain, could have contributed to the anticonvulsant a c t i v i t y of (E,E)-2,3'-diene VPA observed in mice (Abbott and Acheampong, 1985). The concentrations of these metabolites in the various brain regions were a l l below 1 ug/mL, and any s i g n i f i c a n t c l i n i c a l effects a r i s i n g from these products would be highly u n l i k e l y . No anticonvulsant a c t i v i t y was observed in rats when VPA or (E)-2-ene VPA brain concentrations were below 1 ug/mL (Loscher et a/., 1988). 4.9 A N T I C O N V U L S A N T E V A L U A T I O N O F V P A , ( E ) - 2 - E N E V P A , ( E , E ) - 2 , 3 ' - D I E N E V P A , A N D ( E , Z ) - 2 , 3 ' - D I E N E V P A I N R A T S The anticonvulsant a c t i v i t i e s of VPA and (E)-2-ene VPA have been evaluated in mouse. The potency for (E)-2-ene VPA was found to be 50 -100% that of VPA against pentylenetetrazole (PTZ)-induced seizures (Loscher, 1981; Loscher et a/., 1984; Loscher and Nau, 1985; Abbott and Acheampong, 1988). In the current study the anticonvulsant a c t i v i t i e s of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA i n r a t s were evaluated using the PTZ-induced s e i z u r e t e s t . However, u n l i k e previous studies i n which PTZ was i n j e c t e d 15 minutes f o l l o w i n g the a d m i n i s t r a t i o n of the anticonvulsant compound, i n our study the convulsant was administered at the time when b r a i n concentrations of the anticonvulsant were at t h e i r h i g h e s t . The anticonvulsant potencies of VPA and (E)-2-ene VPA i n r a t s were found to be comparable as no s i g n i f i c a n t d i f f e r e n c e s i n t h e i r ED50 values of 1.1 and 1.3 mmol/kg, r e s p e c t i v e l y were observed ( t a b l e 15). S i m i l a r r e s u l t s were noted from the dose-response curves f o r VPA and (E)-2-ene VPA ( f i g u r e 54). These f i n d i n g s were i n agreement with Loscher's observation of (E)-2-ene VPA a c t i v i t y i n mice. The dose-response curve f o r ( E , E ) - 2 , 3 ' - d i e n e VPA shows that potency and i n t r i n s i c a c t i v i t y were l e s s than that of VPA and (E)-2-ene VPA ( f i g u r e 54). An ED50 value of 1.9 mmol/kg was obtained f o r (E ,E)-2 , 3 ' - d i e n e VPA which was s i g n i f i c a n t l y l e s s than that of VPA or (E)-2-ene VPA ( t a b l e 15). The r e s u l t s f o r ( E , E ) - 2 , 3 ' - d i e n e VPA were not i n keeping with previous f i n d i n g s of Abbott and Acheampong (1988) where the potency of the diene i n mice was reported to be comparable to that of (E)-2-ene VPA and 50% that of VPA. The c o n t r a s t i n g r e s u l t s might be due to several reasons. F i r s t l y , d i f f e r e n t species were used to evaluate the a n t i c o n v u l s a n t a c t i v i t y of these compounds. Secondly, the a d m i n i s t r a t i o n of the convulsant 15 minutes a f t e r the a d m i n i s t r a t i o n of 2 , 3 ' - d i e n e VPA i n mice may not have corresponded to peak b r a i n l e v e l s of the d i e n e . F i n a l l y , the anticonvulsant t e s t i n g of the diene by Abbott and Acheampong (1988) used a s o l u t i o n containing a mixture of (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n a 2 . 5 : 1 r a t i o . In the current study a s o l u t i o n c o n t a i n i n g 97% ( E , E ) - 2 , 3 ' - d i e n e VPA was employed. Perhaps the a c t i v i t y observed from the mixture of (E ,E)- and ( E , Z ) - 2 , 3 ' - d i e n e VPA i n the mouse was mainly a t t r i b u t e d to the (E .Z)-isomer. Evidence obtained from t h i s study supports t h i s c l a i m . Following the a d m i n i s t r a t i o n of VPA to r a t s , the only diene detected i n brain was ( E , Z ) - 2 , 3 ' - d i e n e VPA ( f i g u r e 3 8 ) . Therefore, based on these f i n d i n g s the ( E , Z ) - 2 , 3 ' - d i e n e VPA was synthesized and evaluated f o r i t s anticonvulsant a c t i v i t y . The p u r i t y of the sample t e s t e d was found to be 95% the ( E , Z ) - i s o m e r . Using i d e n t i c a l t e s t i n g procedures, the anticonvulsant a c t i v i t y of ( E , Z ) - 2 , 3 ' - d i e n e VPA was found to be s i g n i f i c a n t l y more potent against PTZ-induced s e i z u r e s than ( E , E ) - 2 , 3 ' - d i e n e VPA ( f i g u r e 54). An ED50 value of 1.2 mmol/kg f o r the ( E , Z ) - 2 , 3 ' - d i e n e VPA was comparable to both VPA and (E)-2-ene VPA, and s i g n i f i c a n t l y l e s s than that of ( E , E ) - 2 , 3 ' -diene VPA. I t appears then that the reported anticonvulsant a c t i v i t y i n mice f o r a mixture containing the two diene isomers may have l a r g e l y been due to ( E , Z ) - 2 , 3 ' - d i e n e VPA. Following the a d m i n i s t r a t i o n of (E)-2-ene VPA to r a t s , a marked sedative e f f e c t e d was noted. S i m i l a r CNS e f f e c t s were a l s o observed i n mice (Loscher et a 7 . , 1984; Abbott and Acheampong, 1988). Since the (E)-2-ene VPA i s c u r r e n t l y being i n v e s t i g a t e d as a p o s s i b l e novel anticonvulsant agent f o r i t s apparent l a c k of h e p a t o t o x i c i t y and embryotoxicity i n mice, a second major foreseeable drawback with t h i s compound i s the undesirable CNS e f f e c t s . As stated p r e v i o u s l y , the presence of s i g n i f i c a n t q u a n t i t i e s of VPA and 4-ene VPA i n l i v e r f o l l o w i n g (E)-2-ene VPA a d m i n i s t r a t i o n brings to question the apparent non-hepatotoxic nature of t h i s compound. The use of ( E , E ) - 2 , 3 ' - d i e n e VPA as a s u b s t i t u t e anticonvulsant agent to VPA has a l s o been suggested (Abbott and Acheampong, 1988). However, the current r e s u l t s i n d i c a t e that ( E , E ) - 2 , 3 ' - d i e n e VPA i s not as potent as VPA and the diene may produce some s e r i o u s side e f f e c t s . Following ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n at doses of 150 - 400 mg/kg to r a t s , r i g i d i t y of the s k e l e t a l muscles occurred w i t h i n 10 minutes. This t o x i c i t y was c h a r a c t e r i z e d by the hyperextension of the hindlimbs followed by r i g i d i t y of the back muscles. The t o x i c e f f e c t s were t r a n s i e n t as the animal appeared to recover completely w i t h i n 4 hours. The b a s i s f o r t h i s t o x i c i t y i s not understood although i t does appear to resemble strychnine poisoning (Franz, 1980). There appears to be no r e l a t i o n s h i p between the two t o x i c i t i e s . Strychnine acts at the s p i n a l cord l e v e l as a competitive antagonist to the i n h i b i t o r y neurotransmitter g l y c i n e ( C u r t i s et al., 1967), while the pharmacological e f f e c t of VPA i s thought to be associated with the c e n t r a l i n h i b i t o r y neurotransmitter GABA (Godin et al., 1969; MacDonald and Bergey, 1979). The ( E , Z ) - 2 , 3 ' - d i e n e VPA could prove to be a b e t t e r a l t e r n a t i v e to VPA than e i t h e r ( E , E ) - 2 , 3 ' - d i e n e VPA or (E)-2-ene VPA. The ( E , Z ) - 2 , 3 ' -diene VPA o f f e r s several advantages. No muscle t o x i c i t y was observed f o l l o w i n g the a d m i n i s t r a t i o n of ( E , Z ) - 2 , 3 ' - d i e n e VPA at doses of 75 -300 mg/kg. Nor was there a marked sedative e f f e c t with the ( E , Z ) - 2 , 3 ' -diene VPA. Anticonvulsant a c t i v i t y of the diene was comparable to that of VPA and (E)-2-ene VPA against PTZ-induced s e i z u r e s . F i n a l l y , the ( E , Z ) - 2 , 3 ' - d i e n e VPA may not be hepatotoxic as no 4-ene VPA was detected i n plasma or l i v e r f o l l o w i n g diene a d m i n i s t r a t i o n . I f the metabolites observed f o r ( E , Z ) - 2 , 3 ' - d i e n e VPA are s i m i l a r to those of the (E ,E)-isomer, the p o t e n t i a l f o r h e p a t o t o x i c i t y should be minimized. Future s t u d i e s should determine the pharmacokinetics, t i s s u e d i s t r i b u t i o n , and metabolism of ( E , Z ) - 2 , 3 ' - d i e n e VPA i n r a t s and compare the data to the current study. Favorable r e s u l t s may e v e n t u a l l y lead to the c l i n i c a l use of ( E , Z ) - 2 , 3 ' - d i e n e VPA as an e f f e c t i v e a n t i c o n v u l s a n t . 5 . SUMMARY AND CONCLUSIONS The synthesis of three unsaturated metabolites of VPA, (E)-2-ene VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, and ( E , Z ) - 2 , 3 ' - d i e n e VPA, was s u c c e s s f u l l y completed. The procedure required a d d i t i o n of an e l e c t r o p h i l e to an e s t e r enolate followed by n u c l e o p h i l i c e l i m i n a t i o n of a mesyl e s t e r . The a l k y l a t i n g step was found to be r e g i o s p e c i f i c and the e l i m i n a t i o n of the mesylate was h i g h l y s t e r e o s e l e c t i v e . N u c l e o p h i l i c e l i m i n a t i o n of the mesylate was i n i t i a l l y accomplished using potassium hydride although c a r e f u l c o n t r o l of r e a c t i o n time and temperature was required to prevent side r e a c t i o n s from o c c u r r i n g . For t h i s reason, potassium hydride was replaced with DBU, a reagent much e a s i e r to handle and which gave comparable i f not b e t t e r y i e l d s . Thus, the general s y n t h e t i c procedure was much improved over previous methods. Increased y i e l d s and greater s t e r e o s e l e c t i v i t y were achieved. Isomeric p u r i t i e s of 95 - 97%, as determined by NMR and GC-MS, were attained f o r the synthesized compounds. Several grams of each compound were s y n t h e s i z e d . A negative ion chemical i o n i z a t i o n (NICI) GC-MS a n a l y t i c a l procedure was developed f o r the separation and q u a n t i t a t i o n of VPA and 13 of i t s metabolites extracted from r a t plasma and homogenized t i s s u e samples. The a n a l y s i s of e x t r a c t s of t i s s u e homogenates presented a p a r t i c u l a r problem because column i n t e g r i t y was r a p i d l y compromised. A b a c k - e x t r a c t i o n step together with the use of a pre-column extended the v i a b i l i t y of the column. The NICI assay was much improved, g i v i n g r e s o l u t i o n of the diene metabolites and s e n s i t i v i t y that exceeded previous assays. VPA metabolites could be quantitated r e p r o d u c i b l y i n the picogram range. A f t e r a s i n g l e i . p . dose, the k i n e t i c p r o f i l e of VPA was c h a r a c t e r i z e d i n r a t plasma, l i v e r , kidneys, h e a r t , l u n g s , and i n nine regions of the b r a i n . Enterohepatic r e c y c l i n g of VPA was observed causing a t r a n s i e n t increase i n VPA plasma concentrations 240 minutes f o l l o w i n g the dose. Comparable k i n e t i c p r o f i l e s of VPA were observed between plasma and the peripheral t i s s u e s . Mean concentrations of VPA i n l i v e r throughout the 10 hour time period exceeded that i n plasma with a tissue/plasma r a t i o of 4 . 6 , determined at 10 hours f o l l o w i n g the dose. This may have s i g n i f i c a n c e to the p o t e n t i a l of VPA to be hepatotoxic. S e l e c t i v e binding of VPA to kidney t i s s u e was a l s o observed although t h i s not d i d not occur to the same extent as found i n l i v e r . No evidence was found f o r s e l e c t i v e binding of VPA i n the b r a i n regions examined nor d i d the drug p e r s i s t i n whole b r a i n . S i m i l a r t i s s u e p r o f i l e s were determined f o r (E)-2-ene VPA f o l l o w i n g s i n g l e dose i . p . a d m i n i s t r a t i o n to r a t s . Enterohepatic r e c y c l i n g of (E)-2-ene VPA i n plasma, s i m i l a r to that of VPA, was observed producing a b r i e f increase i n drug concentration 240 minutes f o l l o w i n g a d m i n i s t r a t i o n . The (E)-2-ene VPA appeared to p e r s i s t i n plasma f o r a longer period of time compared to that of VPA. This was a t t r i b u t e d to d i f f e r e n c e s i n plasma p r o t e i n binding as (E)-2-ene VPA i s h i g h l y p r o t e i n bound compared to VPA. The p e r s i s t e n c e of (E)-2-ene VPA i n p e r i p h e r a l t i s s u e s and b r a i n was a l s o observed. This was a r e f l e c t i o n of (E)-2-ene VPA concentrations i n plasma since tissue/plasma r a t i o s d i d not at anytime exceed u n i t y . The a f f i n i t y of (E)-2-ene VPA f o r the l i v e r was l e s s evident than f o r VPA but hepatotoxic i m p l i c a t i o n s must a l s o take i n t o account the metabolites that are formed. D i s t r i b u t i o n of (E)-2-ene VPA i n the b r a i n sections analyzed was found to be uniform. The k i n e t i c p r o f i l e s of ( E , E ) - 2 , 3 ' - d i e n e VPA i n plasma, p e r i p h e r a l t i s s u e s , and various b r a i n regions of r a t s were determined f o r the f i r s t t i m e . There was l e s s evidence f o r enterohepatic c y c l i n g of the diene and clearance from plasma, peripheral t i s s u e s , and b r a i n was more r a p i d than VPA and (E)-2-ene VPA. At very low concentrations (<0.02 ug/g), the diene was s e l e c t i v e l y bound to brain regions with the superior c o l l i c u l u s , i n f e r i o r c o l l i c u l u s , and s u b s t a n t i a n i g r a showing r a t i o s to plasma of 0 . 6 , 1 .4 , and 0 . 8 r e s p e c t i v e l y . There appears to be l i t t l e s i g n i f i c a n c e of t h i s property of the diene to the prolonged pharmacological a c t i v i t y observed with VPA. M e t a b o l i t e p r o f i l e s were determined and compared i n plasma, p e r i p h e r a l t i s s u e s , and nine regions of the b r a i n f o r VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA. Two major o b j e c t i v e s of t h i s study were to determine i f pharmacologically a c t i v e metabolites i n r a t s s e l e c t i v e l y bind and l o c a l i z e i n b r a i n , and the degree of formation of p o t e n t i a l l y hepatotoxic m e t a b o l i t e s . Based on the k i n e t i c p r o f i l e s of VPA metabolites i n b r a i n , i t was concluded that metabolites were not being retained i n b r a i n . The only diene detected i n b r a i n f o l l o w i n g VPA a d m i n i s t r a t i o n was ( E , Z ) - 2 , 3 ' -diene VPA, a minor plasma m e t a b o l i t e . The absence of ( E , E ) - 2 , 3 ' - d i e n e VPA from b r a i n suggests an a c t i v e transport mechanism i s involved whereby the diene i s s t e r e o s e l e c t i v e l y being c l e a r e d from b r a i n at a r a t e g r e a t e r than i t s (E ,Z)-isomer. The suspected hepatotoxic metabolite of VPA, ( E ) - 2 , 4 - d i e n e VPA, was not detected i n l i v e r . The absence of ( E ) - 2 , 4 - d i e n e VPA was a t t r i b u t e d to the covalent binding of t h i s diene to l i v e r t i s s u e . The metabolite 4-ene VPA, a precursor to ( E ) - 2 , 4 - d i e n e VPA, was present i n l i v e r . The metabolites observed f o l l o w i n g s i n g l e dose a d m i n i s t r a t i o n of (E)-2-ene VPA i n plasma, peripheral t i s s u e s , and b r a i n were s i m i l a r to those found a f t e r a s i n g l e dose of VPA. The major pathways of (E)-2-ene VPA metabolism i n r a t s were /J-oxidation, r e d u c t i o n , and i s o m e r i z a t i o n . The main metabolites detected were VPA, 3-keto VPA, and 3-ene VPA. Although ( E ) - 2 , 4 - d i e n e VPA was present i n plasma, i t was not detected i n l i v e r . The concentration of 4-ene VPA was higher i n l i v e r a f t e r (E)-2-ene VPA dosing than a f t e r VPA. Based on the known h e p a t o t o x i c i t y of 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA, the (E)-2-ene VPA may not be f r e e of h e p a t o t o x i c i t y . Upon ( E , E ) - 2 , 3 ' - d i e n e VPA a d m i n i s t r a t i o n , the main route of metabolism was via reduction to VPA, (E)-2-ene VPA, and 3-ene VPA. Since VPA was a major metabolite of ( E , E ) - 2 , 3 ' - d i e n e VPA i n b r a i n , the p o s s i b i l i t y of VPA c o n t r i b u t i n g to the a c t i v i t y of t h i s diene was c o n s i d e r e d . Concentrations of VPA throughout the various b r a i n s e c t i o n s were l e s s than 1 ug/mL over the e n t i r e 10 hour p e r i o d , and a c t i v i t y a r i s i n g from such low l e v e l s would be h i g h l y u n l i k e l y . Two metabolites not detected i n plasma, peripheral t i s s u e s , or brain were 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA. This suggests that the diene may have a lower p o t e n t i a l f o r l i v e r t o x i c i t y compared to that of VPA or (E)-2-ene VPA. The anticonvulsant a c t i v i t y of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' -diene were compared i n r a t s using the standardized PTZ-induced s e i z u r e t e s t . The ED50 values obtained from the dose-response curves were used to compare the r e l a t i v e p o t e n c i e s . VPA and (E)-2-ene VPA had comparable ED50 values and were s i g n i f i c a n t l y more potent than ( E , E ) - 2 , 3 ' - d i e n e VPA. The presence of ( E , Z ) - 2 , 3 ' - d i e n e VPA i n b r a i n f o l l o w i n g VPA a d m i n i s t r a t i o n prompted the t e s t i n g of the ( E , Z ) - i s o m e r . The potency of ( E , Z ) - 2 , 3 ' - d i e n e VPA was found to be equivalent to that of VPA and (E)-2-ene VPA. Sedation was a common observation f o r a l l compounds t e s t e d with (E)-2-ene VPA e l i c i t i n g the most severe response. An unusual pharmacological e f f e c t was observed f o r ( E , E ) - 2 , 3 ' - d i e n e VPA which appeared to resemble strychnine t o x i c i t y . Although the e f f e c t was t r a n s i e n t , t h i s response i n man would severely l i m i t the usefulness of t h i s compound. The ( E , Z ) - 2 , 3 ' - d i e n e VPA was f r e e of t h i s muscular e f f e c t once again p o i n t i n g out that there i s considerable s t e r e o s e l e c t i v i t y i n the pharmacological and pharmacokinetic p r o p e r t i e s of these 2 diene isomers. Although premature, the ( E , Z ) - 2 , 3 ' - d i e n e VPA has p o t e n t i a l f o r use as an anticonvulsant agent. Future i n v e s t i g a t i o n should include s i n g l e dose pharmacokinetic, d i s t r i b u t i o n , and metabolic studies i n r a t s , s i m i l a r to that of VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA, i n order to e s t a b l i s h whether t h i s diene i s r e t a i n e d i n b r a i n or i f p o t e n t i a l hepatotoxic metabolites are formed. I f the r e s u l t s are f a v o r a b l e , perhaps m u l t i p l e dose studies might be considered to e s t a b l i s h whether t h i s diene accumulates i n t i s s u e s , p a r t i c u l a r l y the b r a i n . 204 6. 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APPENDICES 228 TIME (MINUTES) Appendix 1: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n hippocampus f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 229 TIME (MINUTES) Appendix 2: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n superior c o l l i c u l u s f o l l o w i n g 150 mg/kg i . p . j a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 ] pooled samples) 230 100.0 - 1 — 10.0 c n c n Z3 O < or: z o o 1.0 0.1 1.0E-2 © VPA o ( E ) - 2 - E N E V P A • (E,E)-2,3'-DIENE VPA o \ . \ — o 0 200 400 600 TIME (MINUTES) Appendix 3 : VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n i n f e r i o r c o l l i c u l u s f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 231 100.0 10.0 CD CD Z5 o < LU o z o o 1.0 — 0.1 — 1.0E-2 o \ © VPA o ( E ) - 2 - E N E V P A • ( E . E ) - 2 , 3 ' - — • DIENE VPA o o \ \ V-\i o-o— 0 200 400 600 TIME (MINUTES) Appendix 4: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e time curves i n cerebellum f o l l o w i n g a d m i n i s t r a t i o n of each compound to r a t s pooled samples) VPA c o n c e n t r a t i o n - ; 150 mg/kg i . p . j (each data point=8 j 232 100.0 - T -10.0 — C7> =5 o < cr 1.0 — LU o z o o 0.1 — 1.0E-2 • VPA o (E ) -2 -ENEVPA • (E,E)-2.3'-DIENE VPA 0 200 400 TIME (MINUTES) 600 Appendix 5: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n o l f a c t o r y bulbs f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 233 TIME (MINUTES) Appendix 6: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n corpus callosum f o l l o w i n g 150 mg/kg i . p . ! a d m i n i s t r a t i o n of each compound to r a t s (each data point=8! pooled samples) 234 1.0E-2 H 1 1 1 0 200 400 600 T I M E ( M I N U T E S ) Appendix 7: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n s u b s t a n t i a n i g r a f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 235 T I M E ( M I N U T E S ) Appendix 8 : VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n medulla f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 236 100.0 10.0 — C7> Z5 o < on 1.0 Ld o z o o 0.1 — 1.0E-2 © VPA o (E ) -2 -ENEVPA H (E,E)-2,3 I-DIENE VPA Ox 0 o \ • \ \ \ \ -o O—— 200 400 600 TIME (MINUTES) Appendix 9: VPA, (E)-2-ene VPA, and ( E , E ) - 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n -time curves i n putamen f o l l o w i n g 150 mg/kg i . p . a d m i n i s t r a t i o n of each compound to r a t s (each data point=8 pooled samples) 237 1000.0 100.0 - -10.0 — cn =3 o c r 1.0 0.1 y 1.0E-2 o o 1.0E-3 — 1.0E-4 - -1.0E-5 A AA -A. • VPA • (E,E)—2,3'—DIENE VPA • (E.Zj—2,3'—DIENE VPA v M - 2 , 4 - D I E N E VPA o ( E ) - 2 - E N E VPA A 3-ENE VPA A 4 -ENE VPA = 1 = — -fi • 0 200 400 T I M E ( M I N U T E S ) 600 Appendix 10: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 238 100.0 -r-10.0 CT> 1.0 — Z5 o o-1 < or: t £ 1.0E-2 — LjJ O z g 1.0E-3 + 1.0E-4 1.0E-5 0 200 • VPA • (E,E) —2,3' —DIENE VPA • (E l Z)-2,3 , -DIENE VPA o ( E ) - 2 - E N E VPA A 3 - E N E VPA a 4 - E N E VPA — A -- 8 --e 400 600 T I M E ( M I N U T E S ) Appendix 11: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 239 cn CT) Z5 o < LY. LU O Appendix 12: ,E ) -2 ,3 ' -D IENE VPA .Z ) -2 ,3 ' -D IENE VPA 100.0 10.0 1.0 0.1 - h 1 .0E-2 - f a g 1 .0E-3 - h 1 .0E-4 - k 1 .0E-5 • VPA • (E v(E)—2,4—DIENE VPA 0 ( E ) - 2 - E N E VPA A 3 - E N E VPA A 4 - E N E VPA 7<£>V Sr.: -v - • a A A ^ A -0 200 400 600 T I M E ( M I N U T E S ) Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 240 1000.0 -r-100.0 10.0 1.0 — 0.1 1.0E-2 ,..o—•— IO A \ —o-~A, • VPA • 4-KETO VPA v3-KETO VPA O 3 - 0 H VPA A 4 -OH VPA A 5 - O H VPA • 2 - P G A \ \ A A \ A--A 0 200 400 600 TIME (MINUTES) Appendix 13: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 241 100.0 - r -• VPA • 4-KETO VPA v 3-KETO VPA O 3 - 0 H VPA A 4-OH VPA a 5-OH VPA • 2-PGA 10.0 1.0 / A 0.1 +.1cP \ / • \ N \ \ Q—-\ "-O 1.0E-2 0 200 400 600 T I M E ( M I N U T E S ) Appendix 14: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 242 1.0E-2 H 1 1 : 1 0 200 400 600 T I M E ( M I N U T E S ) Appendix 15: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 243 Appendix 16: Concentration-time p l o t of VPA i n whole b r a i n f o l l o w i n g the j a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8/time i p o i n t , S.D. o m i t t e d ) . | 244 100.0 - r 10.0 4 -0.1 + ^ 1 - ° CD cn Z5 z o Pj: 1.0E-2 on o z o o 1.0E-3 + 1.0E-4 -4-1.0E-5 4 -1.0E-6 • VPA o (E ) -2 -ENE VPA A 3 -ENEVPA A 4 -ENEVPA • (E,Z)-2,3'-DIENE VPA A A 4o — A . O. A-O - A -O P ^ c - - • -A O * A ' \ / A 0 200 400 T I M E ( M I N U T E S ) 600 Appendix 17: Concentration-time p l o t s of VPA and i t s unsaturated! metabolites i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i - P - o f V P A t o r a t s ( n = 8 P o o l e d samples/time p o i n t ) . 245 C7> Cn Z5 o < cm Ld O z o o Appendix 18: 100.0 - r 10.0 1.0 0.1 1.0E-2 1.0E-3 — 1.0E-4 1.0E-5 • VPA o ( E ) - 2 - E N E VPA A 3-ENE VPA A 4 -ENE VPA • (E,Z) —2,3'—DIENE VPA 2 0O y \ V A / O -• -o-• -— A o 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n s u p e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 - r 10.0 4 -1.0 4 -0.1 1.0E-2 1.0E-3 4 -1.0E-4 1.0E-5 • VPA o ( E ) - 2 - E N E VPA A 3 -ENE VPA A 4 -ENE VPA • (E,Z)-2,3'-DIENE VPA •«,AO •A o • -A O 0 P - - D -• 0 200 400 600 TIME (MINUTES) Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n i n f e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 - r • VPA o ( E ) - 2 - E N E VPA A 3-ENE VPA A 4 - E N E VPA • (E,Z)—2,3'—DIENE VPA 10.0 - h 1.0 + 0.1 A-O -A-•o-•6 1.0E-2 -frj • • -1.0E-3 — 1.0E-4 + A A -1.0E-5 0 200 400 600 TIME (MINUTES) Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n cerebellum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 248 CP cn Z3 O on UJ o z o o 100.0 - r 10.0 — 1.0 — 0.1 — 1.0E-2 1.0E-3 — 1.0E-4 — 1.0E-5 6* o i - A . •O-• on I • • VPA o ( E ) - 2 - E N E VPA A 3 -ENE VPA A 4 -ENE VPA • (E,Z)-2,3 ,-DIENE VPA -A-O - — A -• O- A 0 200 400 TIME (MINUTES) 600 Appendix 21: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 249 100.0 - i -10.0 CD CD 15 o 1.0 0.1 — < (Y. Ld O 1.0E-2 g 1.0E-3 1.0E-4 1.0E-5 • VPA o ( E ) - 2 - E N E VPA A 3 - E N E VPA A 4 - E N E VPA • (E,Z)-2,3 ,-DIENE VPA —A. o / * • o. A • / • -A-O - • 0 200 400 600 T I M E ( M I N U T E S ) Appendix 22: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n corpus callosum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 250 100.0 io.o 4-CD 15 o !< or: 1.0 4 -LU O z o o 0.1 1.0E-2 1.0E-3 + 1.0E-4 1.0E-5 ^ - A . A • O do -A-O / A I • VPA o(E)—2—ENE VPA A 3-ENE VPA A 4 - E N E VPA • (E,Z)—2,3'—DIENE VPA •O--A O A A 0 200 400 TIME (MINUTES) 600 Appendix 23: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n medulla f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 251 100.0 10.0 — CD 1.0 — Z5 o 0.1 < i CY tz 1.0E-2 — LU O z O 1.0E-3 1.0E-4 — 1.0E-5 • VPA o ( E ) - 2 - E N E VPA A 3-ENE VPA A 4 -ENE VPA • (E,Z)-2,3'—DIENE VPA 8 & > o--A-O • --A O it" ' 0 200 400 TIME (MINUTES) 600 Appendix 24: Concentration-time p l o t s of VPA and i t s unsaturated metabolites i n putamen f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 252 C7> cn =5 Z o <Or: O z o o 100.0 • VPA • 4-KETO VPA v 3-KETO VPA o 3-OH VPA A 4 -OH VPA A 5-OH VPA • 2-PGA 10.0 — 1.0 0.1 — 1.0E-2 0 200 400 600 TIME (MINUTES) Appendix 25: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 253 CD CD Z5 z o !< cr Ld O Z o o 100.0 —i— • VPA • 4-KETO VPA v3-KETO VPA O 3 - 0 H VPA a 4 - O H VPA A 5 -OH VPA • 2-PGA 10.0 1.0 -4-0.1 - h 1.0E-2 1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 26: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n . s u p e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg \ i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 254 100.0 - r -CD Z5 o !< Ld o z o o 10.0 1.0 0.1 1.0E-2 • VPA • 4-KETO VPA v3-KETO VPA O 3 - 0 H VPA A 4 - 0 H VPA A 5-OH VPA • 2-PGA TIME (MINUTES) Appendix 27: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n i n f e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . T I M E ( M I N U T E S ) Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n cerebellum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . or VPA to r a t s (n=8 pooled samples/time p o i n t ) . 256 100.0 - r -CD C7> Z5 O !< CY. Ld O Z o o • VPA • 4-KETO VPA v 3-KETO VPA O3 -0H VPA A 4-OH VPA a 5-OH VPA • 2 - P G A 10.0 — 1.0 — 1.0E-2 0.1 — , v 7 - a TIME (MINUTES) Appendix 29: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 257 100.0 - r • VPA • 4-KETO VPA v 3-KETO VPA O 3 - 0 -A 4 - 0 H V 3 A Vf"' • 2 -PGA V CD CD Z5 10.0 o !< on h-z Ld O Z o o 1.0 — 0.1 — ?W • • v 1.0E-2 TIME (MINUTES) Appendix 30: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n corpus callosum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 258 CD CD Z5 o < cn LU o z o o 100.0 • VPA • 4-KETO VPA v 3-KETO VPA O 3 - 0 H VPA A 4-OH VPA A 5 - O H VPA • 2 - P G A 10.0 1.0 0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 31: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n medulla f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA to r a t s (n=8 pooled samples/time p o i n t ) . 259 100.0 - r Z3 o !< on L J J o z o o • VPA • 4-KETO VPA v 3-KETO VPA O 3 - 0 H VPA A 4 - 0 H VPA A 5 - O H VPA • 2-PGA 10.0 - -1.0 •— 0.1 1.0E-2 1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 32: Concentration-time p l o t s of VPA and i t s p o l a r metabolites i n ! putamen f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of VPA 1 to r a t s (n=8 pooled samples/time p o i n t ) . ! 260 cn ZJ O !< cn o z o o 100.0 O (E ) -2 -ENE VPA v ( Z ) - 2 - E N E V P A • (E,E)-2,3'-DIENE VPA • (E,Z)—2,3'—DIENE VPA A 3 -ENEVPA A 4 -ENEVPA • VPA _ -o--o-10.0 1.0 0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 33: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated! m e t a b o l i t e s , and VPA i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n ! of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , ! S.D. omitted f o r c l a r i t y ) . | 261 1 00.0 * | o 10.0 — Z5 o 1.0 < or: 0.1 — O z o o 1.0E-2 1.0E-3 O (E ) -2 -ENE VPA v (Z ) -2 -ENE VPA • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3-ENE VPA • VPA -o W i _ _ _ _ _ — - A v- — -v \ \ \ • 0 200 400 600 TIME (MINUTES) Appendix 34: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 262 100.0 - r 10.0 o V V / \ CD CD Z5 O LY. 1.0 + l_U O z o o 0.1 4 -1.0E-3 QOO o (E ) -2 -ENE VPA v (Z ) -2 -ENE VPA • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3 -ENEVPA • VPA \ \ \ -A" In 1 .OE-2 -4-\ • / ° " X V • „•• 0 200 400 H 600 T I M E ( M I N U T E S ) Appendix 35: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated j m e t a b o l i t e s , and VPA i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of j 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. j omitted f o r c l a r i t y ) . 263 o (E ) -2 -ENEVPA v (Z ) -2 -ENE VPA • 3-KETO VPA • 3-OH VPA 1.0E-2 -| 1 : 1 1 0 200 400 600 T I M E ( M I N U T E S ) Appendix 36: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 264 100.0 - r o 10.0 + cn CP Z3 o 1.0 < cr. UJ o z o o 0.1 + 1.0E-2 1.0E-3 o O V \ \ \ o (E ) -2 -ENEVPA v (Z ) -2 -ENE VPA T 3-KETO VPA • 3-OH VPA A 5-OH VPA 0 200 -O- •o 400 600 TIME (MINUTES) Appendix 37: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 265 100.0 cn Z5 o !< cn o ooo 10.0 1.0 o (E ) -2 -ENEVPA v (Z ) -2 -ENEVPA • 3-KETO VPA • 3-OH VPA A 5-OH VPA 0.1 O O 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 38: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 266 100.0 - r 10.0 4 -Z3 O o z o o 1.0 4 -0.1 + 1.0E-2 + 1.0E-3 o ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A B (E,E)—2,3' —DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3 -ENEVPA • VPA -O a • t \ • • 0 200 400 600 TIME (MINUTES) Appendix 39: Concentration-time m e t a b o l i t e s , and a d m i n i s t r a t i o n of (n=8/time p o i n t , S p l o t s of (E)-2-ene VPA, VPA i n whole b r a i n 150 mg/kg i . p . of (E)-2 ,D. omitted f o r c l a r i t y ) . i t s unsaturated f o l l o w i n g the •ene VPA to r a t s 267 100.00 -r-cn Z5 O c r L J J o z o o O ( E ) - 2 - E N E VPA v ( Z ) - 2 - E N E VPA • 3-KETO VPA • 3-OH VPA 10.00 1.00 — 0.10 0.01 — 1.00E-3 0 200 400 600 TIME (MINUTES) Appendix 40: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n whole b r a i n f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 268 CD cn Z3 O I— < on UJ o z o o 100.0 O ( E ) - 2 - E N E V P A v m - 2 - E N E V P A • (E,E)-2,3'—DIENE VPA • (E,Z)-2,3'-DIENE VPA A 3 -ENE VPA • VPA 10.0 —_ 1.0 0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 41: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 269 C7> cn O < Y-Z L d O z o o 100.0 ' E ) - 2 - E N E VPA ;Z")-2-ENE VPA :)-2-\ 'E,E)-2,3 , -DIENE VPA >E,Z)-2,3'-DIENE VPA 1 -ENEVPA VPA 10.0 — 1.0 0.1 1.0E-2 — 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 42: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n s u p e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 270 cn =3 o !< cn LU o z o o 100.0 - r o v • • A E ) - 2 - E N E VPA Z J - 2 - E N E VPA E,E)-2,3'-DIENE VPA E,Z)-2.3'-DIENE VPA ENE VPA VPA 10.0 4 -1.0 0.1 1 . 0 E - 2 1.0E-3 0 200 4-00 TIME (MINUTES) 600 Appendix 43: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n i n f e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 271 CD CD Z5 o < cr LU O z o o 100.0 - r -10.0 1.0 0.1 - h 1 .0E-2 4- B o ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • (E,E)—2,3'—DIENE VPA • (E ,Z ) -2 ,3 ' -D IENE VPA A 3 - E N E VPA • VPA 1 .0E-3 0 200 400 TIME (MINUTES) 600 Appendix 44: Concentration-time plots of (E)-2-ene VPA, i t s unsaturated! metabolites, and VPA in cerebellum following the administration of 150 mg/kg i.p. of (E)-2-ene VPA to rats (n=8 pooled samples/time point). 272 100.0 cn cn Z3 O or: h-z Ld O Z o o o ( E ) - 2 - E N E V P A v ( z l - 2 - E N E V P A • (E ,E) -2 ,3 ' -D IENE VPA • (E ,Z) -2 .3 ' -D IENE VPA A 3 - E N E VPA • VPA 10.0 +v v x 1 .0E-3 1 .0E-2 -4-0 200 400 TIME (MINUTES) 600 Appendix 4 5 : , Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 273 cn Z5 O < LU O z o o 100.0 - r 10.0 4 7 7 1.0 4 -0.1 1 .0E-2 4-1 .0E-3 o ( E ) - 2 - E N E V P A v ( z l - 2 - E N E V P A • (E ,E) -2 ,3 ' -D IENE VPA • (E,Z)—2,3'—DIENE VPA A 3 - E N E V P A • VPA \ •v- —v - V — A • • , • — -0 200 400 TIME (MINUTES) 600 Appendix 46: Concentration-time plots of (E)-2-ene VPA, i ts unsaturated metabolites, and VPA in corpus callosum following the administration of 150 mg/kg i .p . of (E)-2-ene VPA to rats (n=8 pooled samples/time point). 274 cn cn z o < or: Ld O z o o 100.0 - r o ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • (E,E)-2,3'-DIENE VPA • (E.Z)—2,3'—DIENE VPA A 3 -ENE VPA • VPA 0.1 -4-\ ,v — \ \ \ \ A 1 .OE-2 + d ° / A-< / 1.0E-3 0 200 4-00 TIME (MINUTES) 600 Appendix 47: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n medulla f o l l o w i n g the a d m i n i s t r a t i o n , of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 p o o l e d ; samples/time p o i n t ) . 275 cn cn Z5 O I— < or I— o z o o 100.0 - r - O ( E ) - 2 - E N E V P A v ( z l - 2 - E N E V P A • (E,E)-2,3 ,-DIENE VPA • (E,Z)—2,3'-DIENE VPA A 3 -ENEVPA • VPA 10.0 1.0 — 0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 48: Concentration-time p l o t s of (E)-2-ene VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n putamen f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled i samples/time p o i n t ) . 276 cn Z o cr h-z LU O z o o 100.0 - r o 10.0 - b 1.0 4-O ( E ) - 2 - E N E V P A v M - 2 - E N E VPA T 3-KETO VPA • 3-OH VPA A 4 -OH VPA A 5-OH VPA 0.1 -4-1.0E-2 1.0E-3 -) 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 49: Concentration-time p l o t s of (E)-2-ene VPA and i t s polar metabolites i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 1.0E-3 H | 1 1 0 200 400 600 TIME (MINUTES) Appendix 50: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n superior c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 cr> Z5 O cn Ld o z o o 10.0 — 1.0 o ( E ) - 2 - E N E V P A v ( Z J - 2 - E N E V P A • 3-KETO VPA • 3-OH VPA A 4-OH VPA A 5-OH VPA 0.1 —_ 1.0E-2 1.0E-3 -j = 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 51: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n i n f e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 279 100.0 CD cn Z5 z o !< cr LU o z o o 10.0 1.0 + 0.1 (E) -2 -ENE VPA M - 2 - E N E VPA 3-KETO VPA 3 - OH VPA 4 - OH VPA 5 - OH VPA 1.0E-2 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 52: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r I metabolites i n cerebellum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time \ p o i n t ) . 280 cn cn Z5 O or: L x J O z o o 100.0 - r O ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • 3-KETO, VPA • 3-OH VPA A 4 -OH VPA A 5-OH VPA 10.0 1.0 0.1 - h 1.0E-2 -4-1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 53: Concentration-time p l o t s of (E)-2-ene VPA and i t s p o l a r metabolites i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 281 cn cr> •Z3 Z o < cn L U o z o o 100.0 - r O ( E ) - 2 - E N E V P A v (Z1-2 -ENEVPA • 3-KETO VPA • 3 -OH VPA A 4 -OH VPA A 5 -OH VPA 10.0 1.0 0.1 1.0E-2 1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 54: Concentration-time p l o t s of (E)-2-ene VPA and i t s polar j metabolites i n corpus callosum f o l l o w i n g the a d m i n i s t r a t i o n of I 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled \ samples/time p o i n t ) . 282 cn cn Z5 O !< cr Ld o z o o 100.0 - r -10.0 4-1.0 — 0.1 - h 1.0E-2 1.0E-3 O v E ) - 2 - E N E VPA Z ) - 2 - E N E VPA • 3-KETO VPA • 3 - 0 H VPA A 4 - 0 H VPA A 5 - 0 H VPA 0 200 400 TIME (MINUTES) 600 Appendix 55: Concentration-time p l o t s of (E)-2-ene VPA and i t s polar metabolites i n medulla f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 283 CD Z5 o i t cr L U o z o o 100.0 - r 10.0 1.0 0.1 + 1.0E-2 1.0E-3 O ( E ) - 2 - E N E V P A v ( Z ) - 2 - E N E V P A • 3-KETO VPA • 3-OH VPA A 4 -OH VPA A 5-OH VPA 1 V V * — A \ • — — 0 200 400 TIME (MINUTES) o -T V * A 600 Appendix 56: Concentration-time p l o t s of (E)-2-ene VPA and i t s polar metabolites i n putamen f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of (E)-2-ene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 284 1.0E-2 H j 1 1 0 200 400 600 TIME (MINUTES) Appendix 57: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 285 1.0E-2 -\ 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 58: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 286 100.0 • (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA A DIENE o (E ) -2 -ENEVPA A 3-ENE VPA • VPA 10.0 cn Z5 O < LU O Z o o 1.0E-2 1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 59: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 287 100.0 - r • (E,E)—2,3'—DIENE VPA • (E,Z)—2,3'—DIENE VPA • 3-KETO VPA • 3-OH VPA v 5-OH VPA 10.0 4-cn CD Z5 o < cn o z o o 1.0 4-0.1 1.0E-2 0 200 400 TIME (MINUTES) 600 Appendix 60: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s polar metabolites i n kidneys f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 288 100.0 - r 10.0 4 -Z5 o < or: 1.0 L J O z o o 0.1 H-• (E,E)-2,3'-DIENE VPA • (E,Z)-2,3'-DIENE VPA T 3-KETO VPA • 3 - 0 H VPA v 5-OH VPA 1.0E-2 0 200 400 TIME (MINUTES) 600 Appendix 61: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n heart f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 289 • (E,E)-2,3'-DIENE VPA 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 62: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n lungs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 290 TIME (MINUTES) Appendix 63: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n whole brain f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8/time p o i n t , S.D. omitted f o r c l a r i t y ) . 291 100.0 • • o A 2,3'-DIENE VPA 2,3'-DIENE VPA E.E E,Z DIENE (E) -2 -ENE VPA 3-ENE VPA VPA Appendix 64: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 292 C7> Z3 o !< cn LU o z o o • (E,E)-2,3'-• (E ,Z)-2 ,3' -100.0 -DIENE VPA -DIENE VPA DIENE O (E ) -2 -ENE VPA A 3-ENE VPA • VPA 10.0 1.0 - -0.1 1.0E-2 1.0E-3 0 200 400 600 TIME (MINUTES) Appendix 65: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n superior c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' -diene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 293 CD Z5 z o < or: Ld o z o o : ,E}-2 ,3 ' -: ,Z ) -2 .3 ' -100.0 • (E,E)— • (E A DIENE O ( E ) - 2 - E N E V P A A 3-ENE VPA • VPA -DIENE VPA DIENE VPA 10.0 — 1.0 — 0.1 1.0E-2 — 1.0E-3 0 200 400 TIME (MINUTES) 600 Appendix 66: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s i unsaturated m e t a b o l i t e s , and VPA i n i n f e r i o r c o l l i c u l u s i f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - . diene VPA to r a t s (n=8 pooled samples/time p o i n t ) . 294 • (E,E)—2,3'—DIENE VPA • (E,Z)-2,3'-DIENE VPA A DIENE O ( E ) - 2 - E N E V P A 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 67: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n cerebellum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 295 • (E,E)-2,3'—DIENE VPA • (E,Z)—2,3' —DIENE VPA A DIENE O (E ) -2 -ENE VPA 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 68: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 10.0 - -1.0 0.1 - -1.0E-2 1.0E-3 • (E,E)-2,3' • (E,Z)-2,3' A DIENE O ( E ) - 2 - E N E V P A A 3 -ENE VPA • VPA DIENE VPA DIENE VPA • \ 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n corpus callosum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 297 • (E,E) —2,3' —DIENE VPA • (E,Z)-2,3'-DIENE VPA A DIENE O ( E ) - 2 - E N E V P A 1.0E-3 - | | | 1 0 200 400 600 TIME (MINUTES) Appendix 70: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA i t s unsaturated m e t a b o l i t e s , and VPA i n medulla f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 298 100.0 • A o A E,E -2 ,3 E,Z)-2,3 DIENE ( E ) - 2 - E N E VPA 3-ENE VPA VPA DIENE VPA DIENE VPA Appendix 71: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA, i t s unsaturated m e t a b o l i t e s , and VPA i n putamen f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 299 • (E,E)—2,3'—DIENE VPA 100.0 - r - • (E,Z)—2,3' —DIENE VPA • 3-KETO VPA • 3-OH VPA • v 5-OH VPA 1.0E-2 - K ' 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 72: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r j metabolites i n hippocampus f o l l o w i n g the a d m i n i s t r a t i o n of 150 \ mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 300 CT) Z5 z o < or o o o 100.0 - r 10.0 4-1.0 0.1 1.0E-2 -4-1.0E-3 • (E,E)-2,3'—DIENE VPA • (E,Z)—2,3' —DIENE VPA T 3-KETO VPA • 3-OH VPA v 5-OH VPA 0 200 400 600 TIME (MINUTES) Appendix 73: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n superior c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 301 1.0E-3 H 1 1 1 0 200 400 600 TIME (MINUTES) Appendix 74: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n i n f e r i o r c o l l i c u l u s f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 302 • (E,E)—2,3'—DIENE VPA 100.0 - r • (E,Z)-2,3'-DIENEVPA • 1.0E-3 H 1 -1 1 0 200 400 600 TIME (MINUTES) Appendix 75: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n cerebellum f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 'E,E)-2,3 , -DIENE VPA V ,Z)-2,3'-DIENE VPA •KETO VPA 3-OH VPA 5-OH VPA 10.0 4 -1.0 0.1 1.0E-2 1.0E-3 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n o l f a c t o r y bulbs f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 100.0 i o . o 4 -1.0 4-0.1 -+-• (E,E)-2,3'-DIENE VPA • (E,Z)—2,3'—DIENE VPA • 3-KETO VPA • 3-OH VPA v 5-OH VPA 1.0E-2 -4-1.0E-3 0 200 400 TIME (MINUTES) 600 Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r \ metabolites i n corpus callosum f o l l o w i n g the a d m i n i s t r a t i o n of ' 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled j samples/time p o i n t ) . 305 1.0E-3 - f - 1 1 ^ 0 200 400 600 TIME (MINUTES) Appendix 78: Concentration-time p l o t s of ( E , E ) - 2 , 3 ' - d i e n e VPA and i t s p o l a r metabolites i n medulla f o l l o w i n g the a d m i n i s t r a t i o n of 150 mg/kg i . p . of ( E , E ) - 2 , 3 ' - d i e n e VPA to r a t s (n=8 pooled samples/time p o i n t ) . 306 100.0 - r -• (E,E)-2,3'-DIENE VPA • (V,Z)-2,3'-DIENEVPA • 3-KETO VPA • 3-OH VPA v 5-OH VPA 10.0 4 -1.0E-2 -4-1.0E-3 0 200 400 600 TIME (MINUTES) i + o nf »F p\ ? V - d i e n e VPA and i t s polar "sSIS s r a m s w s samples/time p o i n t ) . 

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