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Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: comparative metabolic and… Tang, Wei 1995

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FLUORINATED ANALOGUES AS MECHANISTIC PROBES IN VALPROIC ACID HEPATOTOXICITY: COMPARATIVE METABOLIC AND PHARMACOKINETIC STUDIES by WEI T A N G  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE D E G R E E O F DOCTOR O F PHILOSOPHY in THE FACULTY O F GRADUATE STUDIES Faculty of P h a r m a c e u t i c a l  Sciences  Division of P h a r m a c e u t i c a l  Chemistry  W e accept this thesis a s c o n f o r m i n g to the required s t a n d a r d  T H E UNIVERSITY O F BRITISH C O L U M B I A December 1995  © W e i Tang, 1995  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  and study.  his  or  her  representatives.  permission.  TkojC Mn.cevt'Wc «Jl  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  that the  may be It  publication of this thesis for financial gain shall not  of  requirements  I further agree  thesis for scholarly purposes by  the  S  is  that  an advanced  Library shall make it  permission for extensive  granted  by the  understood be  for  that  allowed without  head  of  my  copying  or  my written  Abstract  A s e r i o u s d r a w b a c k in the u s e of the anticonvulsant agent valproic a c i d ( V P A ) is the drug a s s o c i a t e d liver toxicity 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 frequently a c c o m p a n i e d by n e c r o s i s . T h e main objective of this t h e s i s w a s to test the h y p o t h e s i s that the hepatotoxicity of V P A is d u e to the formation of reactive toxic metabolites. Firstly,  metabolic  activation  of  V P A was  investigated  by  detection  c h a r a c t e r i z a t i o n of drug-related thiol conjugates. C o m b i n e d L C / M S / M S  and  and N M R  e v i d e n c e clearly identified 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I in the bile of rats d o s e d with ( E ) - 2 , 4 - d i e n e V P A w h i c h is s u s p e c t e d to play a key role in V P A hepatotoxicity. Sufficient on-line L C / M S / M S d a t a w e r e obtained to indicate the p r e s e n c e of the N A C g l u c u r o n i d e di-conjugate of (E)-2,4-diene V P A in both rat bile a n d urine.  T h e amount  of biliary 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I w a s 7-fold greater than 5 - G S - 3 - e n e V P A , the s u m of the two metabolites a c c o u n t i n g for 6 . 6 % of the d o s e .  Incubation of 2,4-diene  V P A - g l u c u r o n i d e with G S H in the p r e s e n c e of glutathione S - t r a n s f e r a s e ( G S T ) e n z y m e led  to the formation  of the G S H - g l u c u r o n i d e di-conjugate.  T o the  best of  our  k n o w l e d g e , this is the first r e c o r d e d instance in w h i c h g l u c u r o n i d e formation activates a drug to further conjugate with G S H v i a a M i c h a e l addition reaction. In other experiments, L C / M S / M S a n a l y s i s of bile s a m p l e s c o l l e c t e d from rats d o s e d with 4 - e n e V P A , a n a n a l o g u e of the known hepatotoxicant 4 - p e n t e n o i c a c i d (4PA),  indicated the  p r e s e n c e of the  G S H , cysteinylglycine, cysteine and  c o n j u g a t e s of 4 , 5 - e p o x y V P A a n d (E)-2,4-diene V P A , respectively. biliary thiol c o n j u g a t e s a c c o u n t e d for 5 % of the d o s e .  ii  NAC  Quantitatively, the  T h i s o b s e r v a t i o n is n o v e l for 4-  e n e V P A m e t a b o l i s m in terms of the degradation of G S H c o n j u g a t e s p o s s i b l y occurring within the liver a s o p p o s e d to a n inter-organ p r o c e s s w h i c h i n v o l v e s the kidney.  The  G S H - a n d N A C - g l u c u r o n i d e di-conjugates of (E)-2,4-diene V P A w e r e a l s o identified a s metabolites with 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I representing 0 . 0 3 % of the 4 - e n e V P A d o s e . T a k e n together, t h e s e d a t a clearly indicate that reactive metabolites of V P A c a n react with hepatic G S H v i a s e v e r a l different metabolic p a t h w a y s , the s u b s e q u e n t depletion of G S H having potential toxic c o n s e q u e n c e s .  Additionally, (E)-2,4-diene  V P A , in its esterified forms, w a s demonstrated to be c a p a b l e of alkylating r e d u c e d oxytocin at the free c y s t e i n e r e s i d u e s , implicating a direct modification of critical proteins by the d i e n e metabolite of V P A . The  role of G S T in the conjugation  of G S H with ( E ) - 2 , 4 - d i e n e V P A w a s  investigated u s i n g rat liver s u b c e l l u l a r fractions a s the s o u r c e of G S T e n z y m e s . GST  mediated  conjugation  thioester, a structural  of  G S H with  (E)-2,4-diene  The  V P A A/-acetylcysteamine  mimic of the c o r r e s p o n d i n g C o A thioester,  resulted in  two  i s o m e r i c products v i a either 5,6- or 1,6-addition, in a g r e e m e n t with in vivo o b s e r v a t i o n s . O n l y the 1,6-addition product w a s found for the s p o n t a n e o u s reaction of G S H with the u n s a t u r a t e d thioester (control). Quantitatively, G S H conjugates formed in the p r e s e n c e of the c y t o s o l a n d s o n i c disrupted mitoplasts w e r e 2 3 - a n d 2-fold that of control, respectively. N o reaction c o u l d be detected upon a mix of G S H with the free a c i d form of ( E ) - 2 , 4 - d i e n e V P A . T h e results indicate that G S T e n z y m e s e n h a n c e the addition of G S H to (E)-2,4-diene V P A with the esterified d i e n e being e s s e n t i a l for the reaction. T o further e x a m i n e the metabolic activation h y p o t h e s i s , oc-fluoro-4-ene V P A w h i c h w a s e x p e c t e d to be inert to p-oxidative m e t a b o l i s m w a s s y n t h e s i z e d a n d its  iii  effect o n rat liver studied in c o m p a r i s o n with 4 - e n e V P A . F o l l o w i n g treatment of rats for 5 d a y s , 4 - e n e V P A , but not a-fluoro-4-ene V P A , i n d u c e d s e v e r 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 (> 8 5 % affected hepatocytes) a n d alterations in mitochondria. S i m i l a r results were  obtained  when  4-pentenoic  acid  and  2,2-difluoro-4-pentenoic  acid  were  c o m p a r e d . T h e p-oxidation product of 4 - e n e V P A , n a m e l y ( E ) - 2 , 4 - d i e n e V P A , a n d the ^-acetylcysteine  ( N A C ) conjugate  of  the  diene  could  not  be  detected  in  rats  a d m i n i s t e r e d oc-fluoro-4-ene V P A . In a s e p a r a t e acute study, mitochondrial G S H w a s d e t e r m i n e d to remain u n c h a n g e d in rats treated with cc-fluoro-4-ene V P A but w a s r e d u c e d to 6 8 % of control in t h o s e a d m i n i s t e r e d 4 - e n e V P A . T h e s e d a t a are c o n s i s t e n t with results d e r i v e d from metabolic s t u d i e s , s u g g e s t i n g that formation of a reactive intermediate is a key step in the e v e n t s l e a d i n g to 4 - e n e V P A , a n d p o s s i b l y V P A , i n d u c e d liver injury with depletion of mitochondrial G S H a s o n e of the c a u s a t i v e factors. A s u b s e q u e n t investigation w a s c a r r i e d out to c o m p a r e 4 - e n e V P A a n d a-fluoro4 - e n e V P A for their p h a r m a c o k i n e t i c a n d protein binding properties.  The serum  concentration-time profiles of 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A w e r e o b s e r v e d to r e s e m b l e o n e a n o t h e r during the initial 2 0 0 min within w h i c h differences w e r e apparent for the drug effects on mitochondrial G S H . T h e major p h a s e II metabolites w e r e the Lglutamine conjugate for cc-fluoro-4-ene V P A a n d the g l u c u r o n i d e e s t e r for 4 - e n e V P A . T h e toxic metabolite (E)-2,4-diene V P A a n d its N A C conjugate w e r e a g a i n d e t e c t e d only in 4 - e n e V P A treated rats. D e s p i t e differences in m e t a b o l i s m , the disposition to rat liver, the s e r u m p e a k a n d free c o n c e n t r a t i o n s w e r e c o m p a r a b l e for 4 - e n e V P A a n d ocf l u o r o - 4 - e n e V P A . T h u s , the apparent distinction b e t w e e n the two d r u g s in p r o d u c i n g liver toxicity in rats is unlikely to be a s s o c i a t e d with p h a r m a c o k i n e t i c d i f f e r e n c e s .  iv  Finally, b e c a u s e of the apparent nonhepatotoxic property of a-fluoro-4-ene V P A , a-fluoro V P A w a s e v a l u a t e d for anticonvulsant activity in m i c e . T h e E D 5 0 of the drug w a s d e t e r m i n e d to be 1.7 mmol/kg with the p e a k activity occurring at 4 5 - 6 0 min following the d o s e , in contrast to 10 min for V P A . S u b s e q u e n t kinetic s t u d i e s r e v e a l e d that the brain uptake of a-fluoro V P A w a s slower, the p e a k brain c o n c e n t r a t i o n arriving 4 5 min later than in the s e r u m , w h e r e a s the p e a k brain level of V P A c o i n c i d e d with the p e a k s e r u m level occurring within 15 min of the d o s e . O n the other h a n d , a-fluoro V P A a p p e a r e d to persist in the g e n e r a l circulation, resulting in its a p p a r e n t s l o w elimination from the brain. a - F l u o r o V P A w a s d e m o n s t r a t e d to h a v e anticonvulsant activity in the p e n t a m e t h y l e n e t e t r a z o l e s e i z u r e test in mice a n d to be c a p a b l e of i n c r e a s i n g brain s y n a p t o s o m a l G A B A , although the c o n n e c t i o n between t h e s e two e v e n t s r e m a i n s to be clarified.  These  results  suggest  that  a-fluoro  V P A has  potential  anticonvulsant d r u g .  Research Supervisor  F r a n k S . Abbott  as  a  new  Table of Contents Abstract  ii  T a b l e of C o n t e n t s  vi  List of F i g u r e s  ix  List of S c h e m e s  xiii  List of T a b l e s  xv  List of A b b r e v i a t i o n s  xvii  Acknowledgements  xxiii  1.  Introduction  1  1.1.  M e t a b o l i s m of V P A a n d its Metabolites  3  1.2.  V P A Hepatotoxicity  22  1.3.  Use  1.4.  2.  of  Fluorine  Substituted  VPA  Analogues  as  M e c h a n i s t i c P r o b e s in V P A Hepatotoxicity  28  Thesis Objectives  31  Experimental  34  2.1.  Materials  34  2.2.  Instrumentation a n d Analytical M e t h o d s  36  2.3.  Chemical Synthesis  40  2.4.  Determination  of  the  lipophilicity  and  the  ionization  c o n s t a n t s of V P A , 4 - e n e V P A , a-fluoro V P A a n d a-fluoro-4ene V P A 2.5.  59  Detection a n d C h a r a c t e r i z a t i o n of D r u g - A s s o c i a t e d T h i o l C o n j u g a t e s in R a t s T r e a t e d with 4 - E n e V P A or (E)-2,4Diene V P A  2.6.  60  C o n j u g a t i o n of G S H with ( E ) - 2 , 4 - D i e n e V P A C a t a l y z e d by Rat G S T E n z y m e s  68  vi  2.7.  Preliminary  M e t a b o l i c S t u d i e s of a - F l u o r o V P A a n d  a-  F l u o r o - 4 - e n e V P A in R a t s 2.8.  73  C o m p a r a t i v e T o x i c o l o g i c a l S t u d i e s of 4 - E n e V P A a n d a F l u o r o - 4 - e n e V P A in R a t s  2.9.  75  C o m p a r a t i v e P h a r m a c o k i n e t i c a n d M e t a b o l i c S t u d i e s of 4 E n e V P A a n d a - F l u o r o - 4 - e n e V P A in R a t s  2.10.  3.  Comparative  Pharmacokinetic,  Pharmacodynamic  79 and  M e t a b o l i c S t u d i e s of V P A a n d a - F l u o r o V P A in M i c e  86  2.11.  Protein C o n c e n t r a t i o n Determination  91  2.12.  Statistics  91  Results  92  3.1.  Chemical Synthesis  92  3.2.  Ionization C o n s t a n t s a n d the Lipophilicity of V P A , 4 - E n e V P A , a-Fluoro V P A and a-Fluoro-4-ene V P A  3.3.  98  Detection a n d C h a r a c t e r i z a t i o n of Thiol C o n j u g a t e s in R a t s T r e a t e d with 4 - E n e V P A a n d (E)-2,4-Diene V P A  3.4.  99  C o n j u g a t i o n of G S H with ( E ) - 2 , 4 - D i e n e V P A C a t a l y z e d by Rat Liver G S T E n z y m e s  3.5.  Preliminary  Metabolic  133 Studies  of  a-Fluorinated  VPA  A n a l o g u e s in R a t s 3.6.  146  C o m p a r a t i v e T o x i c o l o g i c a l S t u d i e s of 4 - E n e V P A a n d a F l u o r o - 4 - e n e V P A in R a t s  3.7.  154  C o m p a r a t i v e P h a r m a c o k i n e t i c a n d M e t a b o l i c S t u d i e s of 4 E n e V P A a n d a - F l u o r o - 4 - e n e V P A in R a t s  3.8.  Comparative  Pharmacokinetic,  Pharmacodynamic  M e t a b o l i c S t u d i e s of V P A a n d a - F l u o r o V P A in M i c e  vii  169 and 181  4.  Discussion 4.1.  Metabolic  195 Formation  and  Degradation  of  the  GSH  C o n j u g a t e of (E)-2,4-Diene V P A 4.2.  195  E v a l u a t i n g the Ability of ( E ) - 2 , 4 - D i e n e V P A to  Alkylate  P e p t i d e s a n d Proteins 4.3.  211  C o n j u g a t i o n of G S H with (E)-2,4-Diene V P A C a t a l y z e d by Rat Liver G S T E n z y m e s  4.4.  Preliminary  Metabolic  212 Studies  of  a-Fluorinated  VPA  A n a l o g u e s in R a t s 4.5.  Comparative Toxicological  219 S t u d i e s of 4 - E n e V P A a n d  a - F l u o r o - 4 - e n e V P A in R a t s 4.6.  221  C o m p a r a t i v e P h a r m a c o k i n e t i c S t u d i e s of 4 - E n e V P A a n d oc-Fluoro-4-ene V P A in R a t s  4.7.  Comparative  229  Pharmacokinetic,  Pharmacodynamic  Metabolic S t u d i e s of V P A a n d oc-Fluoro V P A in M i c e  and 236  5.  Summary and Conclusions  242  6.  References  246  viii  List of Figures F i g u r e 2 . 1 . P h a r m a c o k i n e t i c m o d e l s for curve-fitting of the s e r u m concentration-time profiles of 4 - e n e V P A a n d a-fluoro-4-ene V P A  85  F i g u r e 2 . 2 . T w o compartmental m o d e l for absorption, distribution a n d elimination of a-fluoro V P A in m o u s e s e r u m a n d brain after a n i.p. d o s e at 0.83 m m o l / k g  90  Figure 3.1. L C / M S / M S m a s s s p e c t r a of (A) 5 - G S - 3 - e n e V P A , (B) 5G S - 2 - e n e V P A , a n d on-line L C / M S / M S detection of (C) 5 - G S - 2 e n e V P A a n d 5 - G S - 3 - e n e V P A in the bile of rats treated with (E)2 , 4 - d i e n e V P A . (D) T h e synthetic s t a n d a r d s s p i k e d in control bile are s h o w n for c o m p a r i s o n  101  F i g u r e 3.2.  M S / M S C I D m a s s s p e c t r a of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e  I o b t a i n e d a s the biliary metabolite in (A) rats treated with (E)-2,4d i e n e V P A a n d (B) a rat d o s e d with [ H ] - 4 - e n e V P A  104  2  7  F i g u r e 3.3.  MS/MS  CID  mass  spectra  of  5-NAC-3-ene  VPA-  g l u c u r o n i d e obtained a s the biliary metabolite in (A) rats treated with (E)-2,4-diene V P A a n d (B) a rat d o s e d with [ H ] - 4 - e n e V P A 2  7  105  F i g u r e 3.4. O n - l i n e L C / M S / M S detection of 5 - N A C - 3 - e n e V P A g l u c u r o n i d e in (A) the bile of rats d o s e d with (E)-2,4-diene V P A , (B) the urine of rats d o s e d with 2,4-diene V P A , a n d (C) the bile of rats d o s e d with [ H ] - 4 - e n e V P A  106  2  7  F i g u r e 3.5. M S / M S m a s s s p e c t r a of the biliary metabolites of (A) 5 - G S 3- e n e V P A - g l u c u r o n i d e II a n d (B) 5 - G S - 2 - e n e V P A - g l u c u r o n i d e in rats treated with (E)-2,4-diene V P A  109  F i g u r e 3.6. (A) H N M R s p e c t r a of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I a n d (B) 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II isolated from the bile of rats d o s e d with (E)-2,4-diene V P A  110  F i g u r e 3.7. M S / M S m a s s s p e c t r a of the biliary metabolites (A) c y s g l y - 4 - h y d r o x y V P A lactone a n d (B) 5 - c y s g l y - 3 - e n e V P A  116  1  5-  F i g u r e 3.8. M S / M S m a s s s p e c t r a of the biliary metabolites (A) 5 - N A C 4- hydroxy V P A lactone a n d (B) 5 - N A C - 3 - e n e V P A  ix  117  F i g u r e 3.9. M S / M S m a s s s p e c t r a of the biliary metabolites (A) 5 - c y s - 4 hydroxy V P A lactone a n d (B) 5 - c y s - 3 - e n e V P A  118  F i g u r e 3.10. O n - l i n e LC/MS/MS detection of 5-GS-3-ene V P A glucuronide in (A) control bile s p i k e d with the r e f e r e n c e c o m p o u n d , (B) the bile of rats treated with 4 - e n e V P A a n d (C) the bile of a rat treated with [ H ] - 4 - e n e V P A  121  2  7  F i g u r e 3 . 1 1 . O n - l i n e L C / M S / M S detection of 5 - N A C - 3 - e n e V P A g l u c u r o n i d e in (A) the bile a n d (B) urine of rats treated with 4 - e n e V P A , a n d (C) the bile of a rat treated with [ H ] - 4 - e n e V P A  122  F i g u r e 3 . 1 2 . O n - l i n e L C / M S / M S detection of 5 - G S - 2 - e n e V P A , 5 - G S - 3 e n e V P A , 5 - G S - 4 - h y d r o x y V P A lactone, 5-cysgly-4-hydroxy V P A lactone a n d 5 - c y s g l y - 3 - e n e V P A in (A) the bile of rats d o s e d with 4 - e n e V P A a n d (B) control bile s p i k e d with the s y n t h e s i z e d standard compounds  124  F i g u r e 3 . 1 3 . O n - l i n e L C / M S / M S detection of 5 - N A C - 4 - h y d r o x y V P A lactone, 5 - N A C - 3 - e n e V P A , 5 - c y s - 4 - h y d r o x y V P A lactone a n d 5c y s - 3 - e n e V P A in (A) the bile a n d (B) urine of rats d o s e d with 4e n e V P A , a n d (C) control bile s p i k e d with the s y n t h e s i z e d standard compounds  125  F i g u r e 3.14. O n - l i n e LC/MS/MS detection of 5-GS-3-ene V P A g l u c u r o n i d e I a n d 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II in (A) after 0 min a n d (B) after 18 h of incubation containing the bile of rats treated with (E)-2,4-diene V P A , a n d (C) after 18 h of incubation of the s a m p l e with p-glucuronidase  128  F i g u r e 3 . 1 5 . (A) L C / M S m a s s s p e c t r u m of a b/s-adduct f o r m e d u p o n reaction of 2 , 4 - d i e n e V P A - N A C A with r e d u c e d oxytocin a n d (B) M S / M S m a s s s p e c t r u m of the b/s-adduct....  131  F i g u r e 3.16. L C / M S m a s s s p e c t r u m of the mixture of products f o r m e d upon reaction of 2,4-diene V P A - g l u c u r o n i d e with r e d u c e d oxytocin  132  F i g u r e 3.17. L C / M S / M S c h r o m a t o g r a m s of (A) 5 - G S - 3 - e n e V P A - N A C A o b t a i n e d from the s p o n t a n e o u s reaction of G S H with 2 , 4 - d i e n e V P A - N A C A , (B) 5 - G S - 2 - e n e V P A - N A C A a n d 5 - G S - 3 - e n e V P A N A C A from the mitoplast promoted reaction, (C) the products from the c y t o s o l promoted reaction, a n d (D) the products from the partially purified G S T c a t a l y z e d reaction  137  2  7  X  F i g u r e 3.18. H N M R s p e c t r a of (A) 5 - G S - 3 - e n e V P A - N A C A a n d (B) 5G S - 2 - e n e V P A - N A C A isolated from the rat liver cytosolic fraction p r o m o t e d conjugation of G S H with 2,4-diene V P A - N A C A  139  F i g u r e 3.19. M S / M S m a s s s p e c t r a of (A) 5 - G S - 3 - e n e V P A - N A C A a n d (B) 5 - G S - 2 - e n e V P A - N A C A isolated from the rat liver c y t o s o l i c fraction promoted conjugation of G S H with 2,4-diene V P A - N A C A . .  140  Figure 3.20. On-line LC/MS/MS detection of 5-GS-3-ene V P A g l u c u r o n i d e I in (A) a q u e o u s solution s p i k e d with the purified metabolites, (B) after 3 0 min of incubation of 2 , 4 - d i e n e V P A g l u c u r o n i d e with G S H in the p r e s e n c e of G S T e n z y m e , (C) after 3 0 min of incubation of 2,4-diene V P A - g l u c u r o n i d e with G S H , (D) after 3 0 min of incubation of 2,4-diene V P A - g l u c u r o n i d e only  145  F i g u r e 3 . 2 1 . G C / M S m a s s s p e c t r a of the t B D M S derivatives of (A) a fluoro V P A a n d (B) a-fluoro-4-ene V P A  149  F i g u r e 3 . 2 2 . G C / M S total ion current c h r o m a t o g r a m of the t B D M S derivatives of a n extracted urine s a m p l e from a V P A treated rat  150  F i g u r e 3 . 2 3 . G C / M S total ion current c h r o m a t o g r a m of the t B D M S d e r i v a t i z e d extract of a urine s a m p l e from a rat treated with a fluoro V P A  151  F i g u r e 3.24. (A) G C / M S total ion current c h r o m a t o g r a m of the T M S d e r i v a t i z e d extract of a urine s a m p l e from a rat treated with a fluoro V P A a n d (B) G C / M S m a s s s p e c t r u m of the T M S derivative of the metabolite a-hydroxy V P A  152  F i g u r e 3 . 2 5 . G C / M S m a s s s p e c t r a of the t B D M S derivatives of urinary (A) a-fluoro V P A - G I n a n d (B) a-fluoro-4-ene V P A - G I n  153  F i g u r e 3.26. P h o t o m i c r o g r a p h s of liver s e c t i o n s from rats a d m i n i s t e r e d (A) 4 - e n e V P A a n d (B) a-fluoro-4-ene V P A , respectively, at 0.70 m m o l / k g daily for 5 d a y s  158  1  F i g u r e 3.27. P h o t o m i c r o g r a p h s of liver s e c t i o n s from rats treated with (A) 4 - P A a n d (B) F 2 - 4 - P A , respectively, at 1.00 mmol/kg daily for 5 days  159  F i g u r e 3.28. Electron micrographs of hepatocytes from a d m i n i s t e r e d (A) 4 - e n e V P A a n d (B) a - f l u o r o - 4 - e n e respectively, at 0.70 mmol/kg daily for 5 d a y s  xi  rats VPA, 160  F i g u r e 3 . 2 9 . G C / M S total ion current c h r o m a t o g r a m s of t B D M S derivatives of extracted urine s a m p l e s from (A) 4 - e n e V P A a n d (B) a-fluoro-4-ene V P A treated rats  166  F i g u r e 3 . 3 0 . (A) G C / M S m a s s s p e c t r u m of t B D M S derivative of the urinary N A C conjugate of (E)-2,4-diene V P A in 4 - e n e V P A treated rats. (B) T h e N A C conjugate of (E)-2,4-diene V P A f o u n d in the urine of 4 - e n e V P A treated rats (C) but not in the urine of a fluoro-4-ene V P A treated rats  167  F i g u r e 3 . 3 1 . M S / M S m a s s s p e c t r u m of the urinary metabolites 2-propyl2-fluoro-5-(A/-acetylcystein-S-yl)-4-hydroxypentanoic a c i d lactone in rats treated with a-fluoro-4-ene V P A at 1.4 m m o l / k g  170  F i g u r e 3 . 3 2 . M S / M S m a s s s p e c t r u m of the urinary metabolites A ^ - ( 2 fluoro-2-propyl-4-pentenoyl)glutamine in rats treated with a fluoro-4-ene V P A at 1.4 m m o l / k g  172  F i g u r e 3 . 3 3 . R e l a t i o n s h i p between the free a n d total c o n c e n t r a t i o n s of 4 - e n e V P A (O) or a-fluoro-4-ene V P A (V) in rat s e r u m  177  F i g u r e 3.34. S e r u m concentration-time profiles of 4 - e n e V P A (O) a n d ( E ) - 2 , 4 - d i e n e V P A (A) in the rats d o s e d i.p. with 4 - e n e V P A at 1.4 mmol/kg  179  F i g u r e 3 . 3 5 . D r u g concentration-time profile of a-fluoro-4-ene V P A in rat s e r u m after a n i.p. injection at 1.4 mmol/kg  180  F i g u r e 3 . 3 6 . L C / M S / M S m a s s s p e c t r u m of the urinary metabolite A ^ - ( 2 fluoro-2-propylpentanoyl)glutamine in m i c e treated with a-fluoro V P A at 0.83 m m o l / k g  183  Figure 3.37A. D r u g concentration-time profiles of a-fluoro V P A in m o u s e brain a n d s e r u m after a n i.p. d o s e of the drug at 0 . 8 3 mmol/kg  188  F i g u r e 3 . 3 7 B . D r u g concentration-time profiles of V P A in m o u s e brain a n d s e r u m a n d its metabolite (E)-2-ene V P A in the s e r u m after a n i.p. d o s e of the drug at 0.83 mmol/kg  189  F i g u r e 3.38. E v a l u a t i o n of the anticonvulsant activities for V P A (V) a n d a-fluoro V P A (O) using the P T Z test  192  xii  List of Schemes S c h e m e 1.1. T h e c h e m i c a l structure of c o - e n z y m e A a n d the pathway of co-enzyme A biosynthesis  2  S c h e m e 1.2. p-Oxidation of fatty a c i d s  6  S c h e m e 1.3. p-Oxidation of V P A  9  S c h e m e 1.4. (A) C y t o c h r o m e P 4 5 0 o x y g e n activation a n d o x y g e n a t i o n , a n d (B) c y t o c h r o m e P 4 5 0 c a t a l y z e d a l k a n e hydroxylation a n d dehydrogenation  11  Scheme  1.5.  Cytochrome  P450  catalyzed  hydroxylation  and  desaturation of V P A  14  S c h e m e 1.6. Glutathione b i o s y n t h e s i s  16  S c h e m e 1.7. M e t a b o l i s m of V P A v i a p-oxidation, P 4 5 0 hydroxylation/dehydrogenation a n d glucuronidation  catalyzed 20  S c h e m e 1.8. M e t a b o l i c p a t h w a y s of V P A l e a d i n g to reactive metabolites a n d their G S H conjugates  21  S c h e m e 1.9. M e t a b o l i c p a t h w a y s e n v i s i o n e d for oc-fluorine-substituted V P A a n a l o g u e s , w h i c h are c h a r a c t e r i z e d by retaining all metabolic p a t h w a y s of V P A except for p-oxidation  32  S c h e m e 3 . 1 . S u m m a r y of the attempted s y n t h e s i s of a - f l u o r o - 3 - e n e V P A a n d a-fluoro-4-ene V P A  93  S c h e m e 3.2. Attempted s y n t h e s i s aminodipropylacetic acid  94  of  a-fluoro  VPA  from  2-  S c h e m e 3.3. P r o p o s e d m e c h a n i s m for the c o n v e r s i o n of a-fluoro-3-ene V P A to 4 - h y d r o x y - 2 - e n e V P A lactone  96  S c h e m e 3.4. S y n t h e s i s of 5 - G S - 3 - e n e V P A from trifluoroethyl 2 - p r o p y l 2,4-pentadienoate  97  S c h e m e 3.5. S y n t h e s i s of 5 - G S - 4 - h y d r o x y V P A lactone  98  xiii  S c h e m e 3.6. Formation of the m/z derivative of a-fluoro V P A  7 7 fragment ion from the t B D M S 147  S c h e m e 4 . 1 . P r o p o s e d a c y l migration of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I to 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II  196  S c h e m e 4 . 2 . S u m m a r y of the metabolic pathways of (E)-2,4-diene V P A leading to the formation of G S H conjugates  203  S c h e m e 4 . 3 . S u m m a r y of the metabolic pathways of 4 - e n e V P A l e a d i n g to the G S H - r e l a t e d m o n o - a n d di-conjugates  210  S c h e m e 4.4. P r o p o s e d reaction m e c h a n i s m s for (A) the G S T c a t a l y z e d conjugation of G S H with 2,4-diene V P A - N A C A to give 5 - G S - 2 e n e V P A - N A C A a n d 5 - G S - 3 - e n e V P A - N A C A , respectively, a n d (B) the s p o n t a n e o u s reaction between G S H a n d 2 , 4 - d i e n e V P A N A C A in a q u e o u s buffer  217  S c h e m e 4 . 5 . M e t a b o l i s m of a-fluoro-4-ene V P A  235  xiv  List of Tables T a b l e 1. C o e f f i c i e n t s of inter-day a n d intra-day variations for a s s a y of biliary mercapturic a c i d pathway metabolites T a b l e 2.  126  T h e G S H related conjugates of 4 , 5 - e p o x y V P A a n d (E)-2,4-  d i e n e V P A e x c r e t e d in the bile of rats treated with 4 - e n e V P A  127  T a b l e 3. G S T activities in rat liver s u b c e l l u l a r fractions  134  T a b l e 4 . C o n j u g a t i o n of G S H with 2,4-diene V P A - N A C A in the p r e s e n c e of rat liver s u b c e l l u l a r fractions  143  T a b l e 5. H i g h resolution m a s s a n a l y s i s of m/z 77 fragment ion d e r i v e d from T M S or t B D M S derivatives of a-fluoro V P A a n d a-fluoro-4ene V P A  148  T a b l e 6. M i c r o v e s i c u l a r s t e a t o s i s in the livers of rats receiving c h r o n i c treatment  156  T a b l e 7. T h e effects of 4 - e n e V P A a n d a-fluoro-4-ene V P A on rat hepatic total glutathione levels  163  T a b l e 8. T h e effects of 4 - e n e V P A a n d a-fluoro-4-ene V P A o n rat liver mitochondrial glutathione levels  164  T a b l e 9. Urinary P h a s e II metabolites of 4 - e n e V P A a n d a - f l u o r o - 4 - e n e VPA  174  T a b l e 10. Inter-assay variations for determination of s e r u m a n d urinary 4 - e n e V P A or a-fluoro-4-ene V P A by G C / M S . . . .  175  T a b l e 1 1 . A p p a r e n t p h a r m a c o k i n e t i c p a r a m e t e r s of 4 - e n e V P A a n d a fluoro-4-ene V P A in rats  181  T a b l e 12. Inter-assay variations for determination of V P A or a-fluoro V P A in the brain, s e r u m or urine by G C / M S  184  T a b l e 13. P h a s e II metabolites of V P A a n d a-fluoro V P A d e t e c t e d in mouse  185  Table  14. M o u s e brain s y n a p t o s o m a l administration of V P A or a-fluoro V P A  XV  GABA  levels  following 193  Table  12. R e l a t i o n s h i p between protection against P T Z - i n d u c e d s e i z u r e s , s y n a p t o s o m a l G A B A levels, a n d brain a-fluoro V P A concentrations  xvi  194  List of Abbreviations AUC  a r e a under s e r u m drug concentration-time curve  BBB  blood-brain-barrier  tBDMSCI  tert-butyldimethylsilyl  BSA  bovine s e r u m albumin  [ H 2  1 8  ]BSTFA  chloride  A/,A/-bis(trimethylsilyl)trifluoroacetamide- H-|8 2  °C  degrees Celsius  calc.  calculated  CDNB  1 -chloro-2,4-dinitrobenzene  CID  collision-induced dissociation  CoA  co-enzyme A  CL.jp  apparent total s e r u m c l e a r a n c e (i.p. d o s e )  CSF  c e r e b r o s p i n a l fluid  5-cys-3-ene V P A  2-propyl-5-(cystein-S-yl)-3-pentenoic a c i d  5 - c y s - 4 - h y d r o x y V P A lactone  2-propyl-5-(cystein-S-yl)-4-hydroxypentanoic a c i d lactone  5-cysgly-3-ene V P A  2-propyl-5-(glycine-cystein-S-yl)-3-pentenoic acid  5 - c y s g l y - 4 - h y d r o x y V P A lactone  2-propyl-5-(glycine-cystein-S-yl)-4hydroxypentanoic a c i d lactone  DBU  1,8-diazabicyclo[5.4.0]undec-7-ene  DCC  dicyclohexylcarbodiimide  XVll  dd  doublet of doublets ( N M R )  2,3'-diene V P A  2-(1'-propenyl)-2-pentenoic a c i d  (E)-2,4-diene V P A  (E)-2-propyl-2,4-pentadienoic a c i d  2,4-diene VPA-glucuronide  1- 0-(2-propyl-2,4-pentadienoyl)-p-Dglucuronide  2,4-diene V P A - N A C A  A/-acetyl-S-((E)-2-propyl-2,4-pentadienoyl) cysteamine  DMAP  4-dimethylaminopyridine  DTNB  5,5'-dithio-b/'s-(nitrobenzoic acid)  DTT  1,4-dithiothreitol  5  c h e m i c a l shift ( N M R )  ED50  d o s e producing given effect in 5 0 % of a n i m a l s  EDTA  ethylenediaminetetraacetic a c i d  El  electron impact ionization  (E)-2-ene V P A  (E)-2-propyl-2-pentenoic a c i d  3 -e n e V P A  2- propyl-3-pentenoic a c i d  4- e n e V P A  2-propyl-4-pentenoic a c i d  ES+  positive e l e c t r o s p r a y  et a l .  et alii  a-fluoro V P A  2-fluoro-2-propylpentanoic a c i d  a-fluoro V P A - G I n  A/ -(2-fluoro-2-propylpentanoyl)glutamine  a-fluoro-4-ene V P A  2-fluoro-2-propyl-4-pentenoic a c i d ;  a-fluoro-4-ene V P A - G I n  A^-(2-fluoro-2-propyl-4-pentenoyl)glutamine  2  xviii  9  gravitational a c c e l e r a t i o n (centrifugation)  GABA  7-aminobutyric a c i d  GABA-T  GABA-oxoglutarate aminotransferase  GAD  glutamate d e c a r b o x y l a s e  GC/MS  combined gas chromatography/mass spectrometry  GR  glutathione r e d u c t a s e  5-GS-2-ene V P A  2-propyl-5-(glutathion-S-yl)-2-pentenoic  acid  5-GS-3-ene V P A  2-propyl-5-(glutathion-S-yl)-3-pentenoic  acid  5-GS-2-ene VPA-glucuronide  1-0-(2-propyl-5-(glutathion-S-yl)-2-pentenoyl)(3-D-glucuronide  5-GS-3-ene VPA-glucuronide I  1- 0-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)P-D-glucuronide  5 - G S - 3 - e n e V P A - g l u c u r o n i d e II  2- 0-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)(3-D-glucuronide  5-GS-2-ene V P A - N A C A  N-acetyl-S-(2-propyl-5-(glutathion-S-yl)-2pentenoyl)cysteamine  5-GS-3-ene V P A - N A C A  A/-acetyl-S-(2-propyl-5-(glutathion-S-yl)-3pentenoyl)cysteamine  5 - G S - 2 - f l u o r o - 4 - h y d r o x y V P A lactone  2-propyl-5-(glutathion-S-yl)-2-fluoro-4hydroxypentanoic a c i d lactone  GSH  glutathione, r e d u c e d form  GSSG  glutathione, o x i d i z e d form  GST  glutathione S-transferase  7-GT  Y-glutamyltranspeptidase  H&E  hematoxylin-eosin  xix  HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfon acid  HMPA  hexamethylphosphoramide  HPLC  high-performance liquid c h r o m a t o g r a p h y  Hz  Hertz ( N M R )  i.p.  intraperitoneal(ly)  i.v.  intravenous(ly)  J  coupling constant in H z ( N M R )  K  apparent elimination rate constant  ka  first order drug absorption rate constant  kio  first order drug elimination rate c o n s t a n t  3-keto-4-ene V P A  2- p r o p y l - 3 - o x o - 4 - p e n t e n o i c a c i d  LC/MS  liquid c h r o m a t o g r a p h y / m a s s s p e c t r o m e t r y  LC/MS/MS  liquid c h r o m a t o g r a p h y / t a n d e m m a s s spectrometry  LDA  lithium d i i s o p r o p y l a m i d e  LDH  lactate d e h y d r o g e n a s e  m  multiplet ( N M R )  MCA  monocarboxylic acid  3-MP  3- m e r c a p t o p r o p i o n i c a c i d  MRM  multiple reaction monitoring  MSTFA  A/-trimethylsilyl-/V-methyltrifluoroacetamide  MTBSTFA  A/-Methyl-A/-(f-butyldimethylsilyl) trifluoroacetamide  XX  m/z  m a s s to c h a r g e ratio ( M S )  NAC  A/-acetyl-L-cysteine  NACA  A/-acety Icysteam i n e  5-NAC-3-ene V P A  2-propyl-5-(A/-acetylcystein-S-yl)-3-pentenoic acid  5-NAC-3-ene VPA-glucuronide  1- 0-(2-propyl-5-(A/-acetylcystein-S-yl)-3pentenoyl)-p-D-glucuronide  5 - N A C - 2 - f l u o r o - 4 - h y d r o x y V P A - l a c t o n e 2- propyl-2-fluoro-5-(A/-acetylcystein-S-yl)-4hydroxylpentanoic a c i d lactone 5-NAC-4-hydroxy VPA-lactone  2-propyl-5-(/V-acetylcystein-S-yl)-4-hydroxyl pentanoic a c i d lactone  NADPH  nicotinamide a d e n i n e dinucleotide p h o s p h a t e reduced  NFSi  A/-fluorobenzenesulfonimide  NMR  n u c l e a r magnetic r e s o n a n c e  P450  cytochrome P 4 5 0  4-PA  4-pentenoic acid  F -4-PA  2,2-difluoro-4-pentenoic a c i d  PB  phenobarbital  PTZ  1,5-pentamethylenetetrazole  q  quartet ( N M R )  r  the coefficient of determination  2  2  s  singlet ( N M R )  s.c.  subcutaneous(ly)  S.D.  s t a n d a r d deviation  xxi  SIM  s i n g l e ion monitoring  t  triplet ( N M R )  t-i/2  half-life  tBDMS  f-butyldimethylsilyl  TCEP  tris-(2-carboxyethyl)phosphine  TFA  trifluoroacetic a c i d  THF  tetrahydrofuran  tR  retention time (chromatography)  v  volume  VPA  valproic a c i d , 2-propylpentanoic a c i d approximately  xxii  Acknowledgements  I s i n c e r e l y thank my r e s e a r c h supervisor, Dr. F. Abbott, for h i s g u i d a n c e a n d support throughout the c o u r s e of this study.  I a m grateful to the other m e m b e r s of my  graduate study committee, Dr. G . Eigendorf, Dr. M . L e v i n e , Dr. R. R e i d a n d Dr. J . Sinclair.  S p e c i a l t h a n k s to Dr. W . R i g g s , Dr. T. Fujimiya, Dr. A . B o r e l , Dr. J . Palaty,  a n d Dr. R. T h i e s , for their constructive s u g g e s t i o n s a n d t e c h n i c a l a s s i s t a n c e . I would a l s o like to thank Mr. M . W e i s s a n d Dr. E. H u m p h r e y (Electron M i c r o s c o p y C e n t e r , U B C ) , M s . L. T r u e m a n a n d Mr. M . lagallo (Department of P a t h o l o g y , U B C ) for s a m p l e preparations for the electron m i c r o s c o p i c a n d light m i c r o s c o p i c e x a m i n a t i o n s .  The  t e c h n i c a l support from Mr. R. Burton in the u s e of G C / M S a n d L C / M S / M S , a n d the c h e m i c a l / a n i m a l s u p p l y a n d other s e r v i c e s from Mr. J . D i b a c y , Mr. M . L a n e , Mr. C . W e i z a n d Mr. D. C h a n are very a p p r e c i a t e d .  Finally, I w o u l d like to a c k n o w l e d g e the  v a r i o u s a s s i s t a n c e from my c o l l e a g u e s , Dr. S . P a n e s a r , M s . J . Z h e n g , M s . S . G o p a u l , M s . J . M o s h e n k o , a n d Mr. A . T a b a t a b e i . T h i s r e s e a r c h w a s s u p p o r t e d by the M e d i c a l R e s e a r c h C o u n c i l of C a n a d a .  xxiii  1. Introduction  V a l p r o i c a c i d ( V P A , 2-propylpentanoic acid) w a s s y n t h e s i z e d in 1 8 8 2 (Burton, 1882).  T h e anticonvulsant activity of V P A w a s d i s c o v e r e d rather fortuitously in 1963  w h e n a group of p h a r m a c o l o g i s t s e m p l o y e d this c o m p o u n d a s a solvent to d i s s o l v e the d r u g s being tested ( C a r r a z et a l . , 1964).  T h e y finally d i s c o v e r e d that the solvent  instead of the d r u g s w a s producing the p h a r m a c o l o g i c a l activity ( C a r r a z et a l . , 1964). V P A w a s m a r k e t e d in F r a n c e in 1967 a n d r e l e a s e d into the United S t a t e s in 1978 for the treatment of v a r i o u s epileptic disorders including a b s e n c e , m y o c l o n i c a n d tonicclonic s e i z u r e s ( B o u r g e o i s , 1989).  Although V P A is r e g a r d e d a s a s a f e drug, it h a s  b e e n found to p r o d u c e potentially fatal liver toxicity ( Z i m m e r m a n a n d Ishak,  1982),  particularly in y o u n g children on polytherapy (Dreifuss et a l . , 1987; 1989), a n d to c a u s e birth d e f e c t s w h e n u s e d during p r e g n a n c y ( N a u et a l . , 1991). T h e f o c u s of this t h e s i s is to investigate the m e c h a n i s m s of V P A hepatotoxicity.  T h u s , a brief review is given  b e l o w to outline the m e t a b o l i s m a n d hepatotoxicity of V P A b e c a u s e the toxicity is generally attributed to biotransformation  of the drug, w h i c h m a y lead to either the  formation of toxic metabolites (Baillie, 1988) or the depletion of hepatic c o - e n z y m e A ( C o A ) (Harris, R. A . et a l . , 1991). A short d i s c u s s i o n about the a d v a n t a g e s of using ocfluorinated  V P A analogues as mechanistic probes  in V P A hepatotoxicity  is a l s o  provided to e m p h a s i z e the utility of t h e s e a n a l o g u e s to discriminate b e t w e e n two major h y p o t h e s e s of V P A hepatotoxicity.  1  NH2  — P—0-P—OH OH OPO3H2  HO  CH2  H3C-C-CH3  O  HO-CH-C-NHCH2CH2CNHCH2CH2SH  Pantothenate kinase —.  Pantothenic acid  ATP  •  4'-Phosphopantothenic acid  ADP  ATP-  Synthetase (L-cysteine)  ADP 4'-Phosphopantothenoyl-L-cysteine  Coenzyme A ADP  N 1  C0 -  Decarboxylase  2  Kinase  ATPDephospho-coenzyme A<  Pyrophosphorylase ^—L^  4'-Phosphopantetheine  ATP  PPi  S c h e m e 1.1. T h e c h e m i c a l structure of c o - e n z y m e A (the upper panel) a n d the pathway of c o - e n z y m e A biosynthesis (the lower panel) ( R o b i s h a w a n d N e e l y , 1985).  2  1.1. Metabolism of VPA and its Metabolites  R e s e a r c h interest in V P A m e t a b o l i s m h a s b e e n stimulated by the h y p o t h e s i s that the rare but fatal hepatotoxicity of V P A results from the formation of reactive, toxic metabolite(s)  (Baillie, 1988; Cotariu a n d Z a i d m a n , 1 9 8 8 ; K a s s a h u n et a l . , 1991).  D e s p i t e its s i m p l e b r a n c h e d c h a i n fatty a c i d structure, the biotransformation of V P A is quite c o m p l e x , including p-oxidation, c y t o c h r o m e P 4 5 0 (P450) c a t a l y z e d oxidation a n d glucuronidation.  1.1.1. p-Oxidation pathway 1.1.1.1.  Co-enzyme A  ( R o b i s h a w a n d N e e l y , 1 9 8 5 ; B r a s s , 1994).  Co-enzyme A  c o n s i s t s of 3 ' - p h o s p h o a d e n o s i n e c o u p l e d through t h e 5' position of the ribose to N(pantothenyl)cysteamine  by a p y r o p h o s p h a t e linkage ( S c h e m e 1.1, u p p e r  panel).  C o m p l e t e b i o s y n t h e s i s of C o A from pantothenate, A T P a n d L-cysteine o c c u r s in cytosol ( S c h e m e 1.1, lower panel). cytosolic  pool,  dephospho-CoA.  or  partially  Mitochondrial C o A m a y b e directly imported from the synthesized  from  either  4'-phosphopantetheine  or  T h e s e two intermediates are taken up by the mitochondria from t h e  cytosolic c o m p a r t m e n t  to a c c o m p l i s h the b i o s y n t h e s i s a n d the resulting  s u b s e q u e n t l y u s e d for v a r i o u s metabolic p r o c e s s e s .  C o A is  A s a n obligate cofactor, C o A  participates in m a n y cellular synthetic a n d oxidative reactions. T h e s e functions include C o A m e d i a t e d p-oxidation of fatty a c i d s , elongation of fatty a c i d s a n d c o m p l e x lipid synthesis.  T h e C o A thioesters of fatty a c i d s a r e a l s o involved in t h e s y n t h e s i s of  3  c h o l e s t e r o l e s t e r s , activation of protein k i n a s e C a n d other regulative a c t i o n s yet to be fully e s t a b l i s h e d . In the liver, the total concentration of cellular C o A (free C o A a n d acyl C o A ) r a n g e s from 2 0 0 to 4 0 0 nmol/g of wet liver (Smith, C . M . , 1978). T h i s concentration is c o n s i d e r e d to be relatively lower than that n e e d e d to a c c o m p l i s h the d i v e r s e roles p l a y e d by C o A ( B r a s s , 1994).  Therefore, it h a s b e e n s u g g e s t e d that a c o n t i n u o u s  recycling of C o A b e t w e e n C o A a n d a c y l - C o A is e s s e n t i a l for maintaining free C o A availability a n d normal cellular functions ( R o b i s h a w a n d N e e l y , 1 9 8 5 ; B r a s s , S u c h a recycling p r o c e s s t a k e s p l a c e between  acyl-CoA  and  L-carnitine  1994). and  is  c a t a l y z e d by carnitine a c y l t r a n s f e r a s e s (Bieber, 1988).  A c y l - C o A + carnitine <-> Acylcarnitine + C o A S H  A l t h o u g h the s y n t h e s i s of C o A c a n be a c c o m p l i s h e d in c y t o s o l , about 7 0 % (rat liver) to 9 5 % (rat heart) of total C o A is located in the mitochondrial matrix ( R o b i s h a w a n d N e e l y , 1985). T h e r e f o r e , a transport s y s t e m must exist to be c a p a b l e of importing C o A into mitochondria against a large concentration gradient 1987).  (Tahiliani a n d N e e l y ,  T h i s a s s u m p t i o n m a y not be n e c e s s a r i l y true if most mitochondrial  C o A is  partially s y n t h e s i z e d from cytosolic intermediates.  1.1.1.2.  p-Oxidation of fatty acids  (Schulz, 1991; G u z m a n and G e e l e n ,  1993).  C o n v e r s i o n of fatty a c i d s to their C o A thioesters is a prerequisite to fatty a c i d poxidation.  In the p r e s e n c e of A T P , a group of e n z y m e s c a l l e d a c y l - C o A s y n t h e t a s e s  c a t a l y z e the coupling of free a c i d s with C o A .  4  A c y l - C o A s y n t h e t a s e s are found both  inside a n d outside the mitochondria a n d fall into three c a t e g o r i e s a c c o r d i n g to their p r e f e r e n c e s for different lengths of fatty a c i d s , n a m e l y short-chain, m e d i u m - c h a i n (in the  mitochondrial  matrix)  and  long-chain  acyl-CoA  synthetases  (in  the  outer  mitochondrial m e m b r a n e , m i c r o s o m e s a n d p e r o x i s o m e s ) . Short ( C 4 - CQ) a n d m e d i u m (CQ - C14)  c h a i n fatty a c i d s m a y be taken up directly by mitochondria a n d activated to  the c o r r e s p o n d i n g C o A thioesters within the matrix.  T h e transport of long (C14  - C-|s)  c h a i n fatty a c i d s into mitochondria, however, requires the a s s i s t a n c e of a carnitine d e p e n d e n t carrier s y s t e m ( S c h e m e 1.2). Once  inside the  mitochondrial  matrix,  fatty a c i d  C o A thioesters  are  the  s u b s t r a t e s of a c y l - C o A d e h y d r o g e n a s e s that are a g a i n g r o u p e d on the b a s i s of their fatty a c i d c h a i n length specificities. T h e reaction p r o c e e d s v i a oc-proton abstraction a n d p-hydride  transfer  to  result  in a n a,p-unsaturated  acyl-CoA  or ( E ) - 2 - e n o y l - C o A .  S u b s e q u e n t l y the ( E ) - 2 - e n o y l - C o A is subjected to hydration c a t a l y z e d by e n o y l - C o A h y d r a t a s e (crotonase) to p r o d u c e 3 - h y d r o x y a c y l - C o A .  F o r a long c h a i n (E)-2-enoyl-  C o A , s u c h a hydration reaction is c a t a l y z e d by a long-chain e n o y l - C o A h y d r a t a s e . T h e third step in the p-oxidative reaction s c h e m e is the d e h y d r o g e n a t i o n of 3 - h y d r o x y a c y l C o A by 3 - h y d r o x y a c y l - C o A d e h y d r o g e n a s e to the product 3 - k e t o a c y l - C o A . Finally, 3k e t o a c y l - C o A is c l e a v e d at the p-position  by 3 - k e t o a c y l - C o A - t h i o l a s e , resulting  in  a c e t y l - C o A a n d a n a c y l - C o A derivative containing two c a r b o n s l e s s t h a n the original a c y l - C o A m o l e c u l e . W h i l e a c e t y l - C o A c a n be o x i d i z e d to c a r b o n dioxide a n d water v i a the citric a c i d c y c l e , the s h o r t e n e d a c y l - C o A re-enters the p-oxidative p a t h w a y to a c h i e v e a c o m p l e t e oxidation of the fatty a c i d ( S c h e m e 1.2).  5  ATP R-CH -CH -C-OH 2  2  A M P + PPj *  7 ^  C o A(- S H  Acyl-CoA synthetase  f?  R-CH -CH -C-S-CoA 2  2  Carnitine-^ CPTI O  Inner mitochondrial membrane  -- R - C H - C H - C - c a r n i t i n e 2  2  CoA—0  CPT  Carnitine-^ O R-CH -CH -C-S-CoA 2  2  Acyl-CoA dehydrogenase F H P  2  R-CH=CH-C-S-CoA  (E)-2-Enoyl-Co/>|p 20 H  hydratase  2  CO2  OH  O  R-CH-CH2-C-S-C0A  Citric acid cycle p-Hydroxyacyl-CoA f dehydrogenase ^  N A D H + H+  Q  0: R-C-S-CoA +  NAD+  11 • II R-CTCH -C-S-COA  CH3-C-S-C0A  2  CoA-SH S c h e m e 1.2. p-Oxidation of fatty a c i d s . flavoprotein ( S c h u l z , 1991).  C P T , carnitine  6  palmitoyltransferases;  F , p  A modified form of p-oxidation o c c u r s in p e r o x i s o m e s , leading to the formation of a c e t y l - C o A a n d h y d r o g e n peroxide ( O s m u n d s e n et a l . , 1991).  W h i l e p e r o x i s o m a l p-  oxidation h a s a b r o a d substrate specificity, it is e s p e c i a l l y active t o w a r d s very long c h a i n fatty a c i d s .  T h e p e r o x i s o m a l p-oxidation products are s u b s e q u e n t l y transferred  to mitochondria for further m e t a b o l i s m .  1.1.1.3.  p-Oxidation of VPA.  a c i d p-oxidation s y s t e m .  V P A proved to be a substrate for the mitochondrial fatty  In studies using purified mitochondrial e n z y m e s (Ito et a l . ,  1990) or isolated rat liver mitochondrial fraction (Bjorge a n d Baillie, 1 9 9 1 ; Li et a l . , 1991) , the drug w a s s h o w n to be c o n v e r t e d to the c o r r e s p o n d i n g C o A thioester a n d w a s then d e h y d r o g e n a t e d by 2 - m e t h y l - b r a n c h e d - c h a i n a c y l - C o A d e h y d r o g e n a s e to (E)2 - p r o p y l - 2 - p e n t e n o y l - C o A ((E)-2-ene V P A - C o A ) .  Further hydration of ( E ) - 2 - e n e V P A -  C o A by e n o y l - C o A h y d r a t a s e (crotonase) results in 2 - p r o p y l - 3 - h y d r o x y p e n t a n o y l - C o A (3-hydroxy V P A - C o A ) . A n incorporation of  1  8  0 into the p-hydroxy a c i d metabolite w a s  o b s e r v e d w h e n incubation of V P A with rat liver mitochondria w a s carried out in  1  8  0-  labelled water, indicating that the s o u r c e of the hydroxy o x y g e n a t o m w a s the a q u e o u s m e d i u m (Bjorge a n d Baillie, 1991). T h e d e h y d r o g e n a t i o n of 3-hydroxy V P A - C o A to 2p r o p y l - 3 - o x o p e n t a n o y l - C o A (3-keto V P A - C o A ) w a s d e m o n s t r a t e d to be c a t a l y z e d by a novel m e m b r a n e - b o u n d N A D + d e p e n d e n t d e h y d r o g e n a s e (Li et a l . , 1991), a n d the resulting 3-keto acid metabolite a p p e a r e d to resist c l e a v a g e by 3 - k e t o a c y l - C o A thiolase (Li et a l . , 1991), leading to a n incomplete p-oxidative p r o c e s s . F r o m a s t e r e o c h e m i c a l point of view, d a t a derived from  rat  hepatocyte cultures containing  [5- C]VPA 1 3  indicated that p-oxidation of V P A exhibited a slight p r e f e r e n c e (~ 1.3 to 1) for attack on the  pro-S propyl  s i d e - c h a i n (Shirley et a l . , 1993). T h i s result w a s in contradiction to the  7  k n o w n fact that oxidation of i s o b u t y r y l - C o A a n d 2 - m e t h y l b u t y r y l - C o A by 2-methylb r a n c h e d - c h a i n a c y l - C o A d e h y d r o g e n a s e is highly s t e r e o s e l e c t i v e (Ikeda a n d T a n a k a , 1988). It is of interest to note that L-carnitine, a cofactor required for the entry of longc h a i n fatty a c i d s into mitochondria (Section 1.1.1.1), is not a requirement for V P A transport b e c a u s e the p-oxidation of V P A w a s o b s e r v e d to be i n d e p e n d e n t of the p r e s e n c e of L-carnitine (Bjorge a n d Baillie, 1991). E v i d e n c e w a s a l s o provided to s u g g e s t that p-oxidation of V P A involves the participation of hepatic p e r o x i s o m e s (van d e n B r a n d e n a n d R o e l s , 1 9 8 5 ; V a m e c q et a l . , 1993).  F o r m a t i o n of h y d r o g e n peroxide, a by-product of p e r o x i s o m a l p-oxidation, w a s  o b s e r v e d to be stimulated upon incubation of V P A with rat liver h o m o g e n a t e ( V a m e c q et a l . , 1993). distributions  T h e activity of a s o - c a l l e d v a l p r o y l - C o A o x i d a s e d i s p l a y e d density (isopycnic  centrifugation  on  a  sucrose  density  gradient)  which  s u p e r i m p o s e d with that of the p e r o x i s o m a l m a r k e r e n z y m e c a t a l a s e rather than with mitochondrial c y t o c h r o m e c o x i d a s e ( V a m e c q et a l . , 1993).  But, unlike mitochondrial  V P A p-oxidation w h e r e V P A metabolites w e r e clearly identified, p e r o x i s o m a l p-oxidation of V P A w a s e s t i m a t e d b a s e d entirely  upon h y d r o g e n p e r o x i d e formation.  The  i n c r e a s e d generation of h y d r o g e n peroxide c o u l d result from V P A i n d u c e d impairment of mitochondrial p-oxidation (Section 1.2), which w o u l d shift e n d o g e n o u s fatty a c i d s to the p e r o x i s o m a l p a t h w a y (van d e n B r a n d e n a n d R o e l s , 1985).  T h e contribution from  p e r o x i s o m e s to the biotransformation of V P A , if any, a p p e a r s to b e m i n i m u m  in  c o m p a r i s o n with mitochondrial p-oxidation (Bjorge a n d Baillie, 1 9 9 1 ; Li et a l . , 1991). The  p-oxidative  metabolism  of  V P A was  shown  to  be  dose  dependent.  F o l l o w i n g a single oral administration of V P A to healthy adults, 3-keto V P A e x c r e t e d in  8  the urine, e x p r e s s e d a s a fraction of the d o s e , w a s 3 3 . 7 % at lower d o s e s (250 mg) but d e c r e a s e d to 2 0 . 7 % w h e n higher d o s e s (1000 mg) w e r e a d m i n i s t e r e d ( G r a n n e m a n et al., 1984a). COOH  (VPA) Inner mitochondrial membrane 2-Methyl-branched -chain a c y l - C o A dehydrogenase  COSCoA  (VPA-CoA ester)  COSCoA  ((E)-2-Ene V P A - C o A ester)  Crotonase  COSCoA  A n unknown m e m b r a n e -bound NAD+ dependent dehydrogenase  COSCoA  OH (3-Keto V P A - C o A ester)  (3-Hydroxy V P A - C o A ester)  S c h e m e 1.3. p-Oxidation of V P A ( J a g e r - R o m a n et a l . , 1 9 8 6 ; Bjorge a n d Baillie, 1 9 9 1 ; Li e t a l . , 1991).  1.1.2. Cytochrome P450 catalyzed oxidation 1.1.2.1. Cytochrome P450 1990).  (Ortiz d e Montellano, 1 9 8 9 ; G u e n g e r i c h a n d M a c D o n a l d ,  C y t o c h r o m e s P 4 5 0 a r e a s p e c i a l c l a s s of h e m e containing  9  multicomponent  e n z y m e s that are widely distributed in m a m m a l i a n , plant a n d microbial s y s t e m s . T h e s e e n z y m e s are s o d e s i g n a t e d b e c a u s e a n a b s o r b a n c e at 4 5 0 nm w a s o b s e r v e d for the c a r b o n m o n o x i d e difference s p e c t r u m of the r e d u c e d e n z y m e s ( O m u r a a n d S a t o , 1964).  The  P 4 5 0 e n z y m e s generally  function  to  insert  an  oxygen  atom  into  e n d o g e n o u s a n d xenobiotic substrates. O x y g e n transfer w a s initially b e l i e v e d to follow a c o n c e r t e d , two-electron m e c h a n i s m : binding of a substrate m o l e c u l e to the e n z y m e a n d o n e electron flow from N A D P H - c y t o c h r o m e P 4 5 0 r e d u c t a s e to P 4 5 0 ; binding of a d i o x y g e n m o l e c u l e to the e n z y m e - s u b s t r a t e c o m p l e x , a s e c o n d electron  transferred  from N A D P H - c y t o c h r o m e P 4 5 0 reductase a n d o x y g e n activation; finally incorporation of an oxygen atom  into the substrate  ( S c h e m e 1.4A).  T h e characteristics of  this  c o n c e r t e d m e c h a n i s m are that the substrate stereochemistry a p p e a r s to b e c o n s e r v e d a n d the reaction provides for low kinetic isotope effects (Ortiz d e M o n t e l l a n o , 1986). M o r e recently, e v i d e n c e w a s obtained to indicate that P 4 5 0 c a t a l y z e d reactions m a y involve free radicals a s fleeting intermediates.  In this n o n - c o n c e r t e d p r o c e s s or s o  c a l l e d free radical m e c h a n i s m , the stereochemistry of the substrate is not c o n s e r v e d and  the  intrinsic  isotope  effects  are  quite  large  (White,  R.  E.  et  al.,  1986).  C o n s e q u e n t l y , s o m e t i m e s a d e h y d r o g e n a t i o n reaction rather than a m o n o - o x y g e n a t i o n reaction is c a t a l y z e d by the P 4 5 0 e n z y m e s a n d this reaction c a n b e  satisfactorily  a c c o u n t e d for by this m e c h a n i s m ( S c h e m e 1.4B) ( G u e n g e r i c h a n d M a c D o n a l d , 1990). A wide variety of drugs or environmental c h e m i c a l s ranging from aliphatic a n d aromatic h y d r o c a r b o n s to heteroatom (oxygen, sulfur a n d nitrogen) containing c o m p o u n d s are s u b s t r a t e s of P 4 5 0 e n z y m e s . T h e hepatic P 4 5 0 e n z y m e s are well r e c o g n i z e d a s the primary e n z y m e s involved in drug m e t a b o l i s m .  10  P  P450[Fe]3  §  +  >  P450[Fe]3  P450[Fe]3+  +  1e-  [P] P450[Fe]2+ [S] P450[FeO]3+ [S]  0  2  H 0 2  2H+  P450[FeO ] 2  P450[FeO2]+ [S]  1e-  1  2  [S]  S c h e m e 1.4A. C y t o c h r o m e P 4 5 0 o x y g e n activation a n d o x y g e n a t i o n . S , substrate; P, product ( G u e n g e r i c h a n d M a c D o n a l d , 1990). OH  I  ^CH CH ' 2  ^ C H  ,CH  2 X  CH "  V  2  P450[FeO]3+  P450[Fe]3+ CH " 2  P450[FeOH]3+  |H^b  N  >  P450[Fe]3+  S c h e m e 1.4B. C y t o c h r o m e P 4 5 0 c a t a l y z e d a l k a n e hydroxylation a n d d e h y d r o g e n a t i o n .  11  T h e P 4 5 0 e n z y m e s are a l s o k n o w n to be inducible, a term u s e d for the e l e v a t e d e x p r e s s i o n of a particular e n z y m e d u e to the perturbation of the p h y s i o l o g i c a l state by x e n o b i o t i c s o r environmental c h a n g e s (Okey, 1990).  Several mechanisms have been  p r o p o s e d for t h e o b s e r v e d induction of P 4 5 0 , including i n c r e a s e d s y n t h e s i s a n d stabilization of t h e e n z y m e proteins (Okey, 1990).  C o n s e q u e n c e s arising from t h e  induction of P 4 5 0 are a s s o c i a t e d with alterations in t h e efficacy of therapeutic a g e n t s a n d i n c r e a s e d toxicities from drugs or environmental c h e m i c a l s (Okey, 1990)  1.1.2.2. Cytochrome P450 catalyzed oxidation of VPA.  In addition to p-oxidation,  m i c r o s o m a l P 4 5 0 m e d i a t e d biotransformation is a major p h a s e I p a t h w a y for V P A , leading to the formation of 2-propyl-4-hydroxypentanoic a c i d (4-hydroxy V P A ) , 2-propyl5 - h y d r o x y p e n t a n o i c a c i d (5-hydroxy V P A ) a n d 2-propyl-4-pentenoic a c i d (4-ene V P A ) (Prickett a n d Baillie, 1984; Rettie et a l . , 1987; 1988). T h e first two metabolites a p p e a r to result from t h e P 4 5 0 c a t a l y z e d incorporation of o x y g e n a t o m s from m o l e c u l e s into t h e V P A substrate.  dioxygen  S u c h a m o n o - o x y g e n a t i o n reaction is thought to  o c c u r v i a h y d r o g e n atom abstraction from either t h e 4 - o r 5-position by t h e P 4 5 0 oxygen  complex  (perferryl  [FeO] ) 3 +  to form  V P A radicals  followed  r e c o m b i n a t i o n a c c o r d i n g to the m e c h a n i s m outlined in S c h e m e 1.5.  by radical  In support of this  radical intermediate m e c h a n i s m , large intramolecular isotope effects w e r e o b s e r v e d for both 4-hydroxylation (k^/k^: 3.7 - > 5) a n d 5-hydroxylation {ky\lktf. 4.4 - 6.4) (Rettie et al., 1988).  Similarly, t h e P 4 5 0 c a t a l y z e d desaturation of V P A to 4 - e n e V P A w a s  p r o p o s e d to involve a n intermediate V P A radical at the 4-position, {k^lk^ 3.6 - 7.6) that, i n s t e a d of being terminated by recombination, underwent l o s s of a n electron to t h e P450-oxygen complex.  T h e resulting c a r b o n i u m ion l o s e s a s e c o n d proton at the 5 -  12  position with the formation of the final unsaturated product ( S c h e m e 1.5) (Rettie et a l . , 1988).  Further oxidation of 4-hydroxy V P A results in 2 - p r o p y l - 4 - o x o p e n t a n o i c a c i d (4-  keto V P A ) a n d 2 - p r o p y l s u c c i n i c a c i d ( P S A ) , while oxidation of 5-hydroxy V P A p r o d u c e s 2-propylglutaric a c i d ( P G A ) ( A c h e a m p o n g et a l . , 1 9 8 3 ; Baillie a n d R e t t e n m e i e r , 1989). R e c e n t l y , a n investigation using rabbit lung m i c r o s o m e s a s a rich s o u r c e of CYP4B1  or c D N A  expressed C Y P 4 B 1  indicated that this i s o e n z y m e w a s  mainly  r e s p o n s i b l e for the m i c r o s o m a l desaturation of V P A (Rettie et a l . , 1995). T h e partition b e t w e e n o x y g e n r e b o u n d a n d desaturation at the C - 4 position of the V P A radical w a s 2 to 1 (Rettie et a l . , 1995).  In other w o r d s , generation of e v e r y two m o l e c u l e s of 4-  hydroxy V P A from V P A is a c c o m p a n i e d by the formation of o n e m o l e c u l e of 4 - e n e V P A . T h i s terminal unsaturated V P A metabolite is known to i n d u c e hepatotoxicity in rats (Section 1.2). T h e rat P 4 5 0 c a t a l y z e d hydroxylation of V P A w a s o b s e r v e d to result in 3hydroxy V P A (Rettie et a l . , 1987).  A l t h o u g h this (3-hydroxy a c i d is b e l i e v e d to be  involved in the p-oxidative chain reactions (Bjorge a n d Baillie, 1 9 9 1 ; Li et a l . , 1991), only t r a c e a m o u n t s of the metabolite w e r e detected in the rat liver  mitochondrial  incubations with V P A (Li et a l . , 1991). F o r m a t i o n of 3-hydroxy V P A from 2 - [ H ] V P A in 2  vivo in rats retained 7 2 % of the label (Rettenmeier et a l . , 1987). V P A with rat hepatic m i c r o s o m a l fraction under incorporation of o n e atom of  1  8  0  1  8  0  In vitro incubation of  o x y g e n a t m o s p h e r e led to the  into the p-hydroxy a c i d metabolite (Prickett a n d  Baillie, 1984). T h e s e d a t a s u g g e s t that most of the 3-hydroxy V P A found in the urine of rats or patients treated with V P A is of m i c r o s o m a l ( e n d o p l a s m i c reticulum) origin.  13  COOH  [FeOH]  [Fe] *  2+  OOOH  3  J O  [FeOHf\. "  (3-Ene VPA)  [FeOH] V 3  COOH  [FeOHf  [Fe]  +  OH (3-Hydroxy VPA)  [FeOH],3+  [Feof  C  O  O  [FeOH]  H  3+  [FeO]  +  3+  COOH  3+  y COOH (VPA) ,3+ -[FeO]  [FeOH] " " 3  1  [Fe] ,3+  [FeOH]',3+  COOH COOH HO  [FeOH]  [Fe]  3+  3+  o. ,[FeOHr  (5-Hydroxy VPA)  HO  COOH  (4-Hydroxy VPA)  +  J X [FeOH] " 24  COOH  [FeOH]  [Fe]  2+  H 0 2  3+  COOH  (4-Ene VPA)  S c h e m e 1.5. C y t o c h r o m e P 4 5 0 c a t a l y z e d hydroxylation a n d d e s a t u r a t i o n of V P A (Ortiz d e M o n t e l l a n o , 1 9 8 9 ; Rettie et a l . , 1995).  14  It is well r e c o g n i z e d that m e t a b o l i s m of V P A v i a the P 4 5 0 m e d i a t e d pathway c a n be affected by c o - m e d i c a t i o n with phenobarbital ( P B ) , phenytoin a n d c a r b a m a z e p i n e ( K a s s a h u n et a l . , 1990; L e v y et a l . , 1990).  E l e v a t e d levels of 4 - e n e V P A w e r e  o b s e r v e d in patients w h o w e r e under polytherapy ( K a s s a h u n et a l . , 1 9 9 0 ; L e v y et a l . , 1990).  R e c e n t l y , both C Y P 2 B a n d C Y P 4 w e r e reported to be i n d u c e d by V P A in rat  h e p a t o c y t e cultures (Akrawi et a l . , 1993). G i v e n the fact that desaturation of V P A to 4e n e V P A is m e d i a t e d by C Y P 2 B a n d C Y P 4 B families (Rettie et a l . , 1995), s u c h a n o b s e r v a t i o n m a y h a v e significance to V P A therapy.  1.1.3. The glutathione (GSH) dependent pathway 1.1.3.1. GSH and GSH conjugation  ( R e e d , 1990; D e L e v e a n d K a p l o w i t z , 1991).  G l u t a t h i o n e is a tripeptide (L-7-glutamyl-L-cysteinyl-glycine) representing the m a i n n o n protein cellular thiol a n d is s y n t h e s i z e d in a two step reaction. T h e first s t e p , c a t a l y z e d by 7-glutamylcysteine s y n t h e t a s e , involves the coupling of L-glutamate with L-cysteine a n d is rate  limiting during  G S H synthesis.  Formation  of  G S H is  subsequently  a c c o m p l i s h e d v i a the G S H s y n t h e t a s e c a t a l y z e d reaction of 7-glutamylcysteine with Lglycine.  T h e overall reaction requires two m o l e c u l e s of A T P , o n e for e a c h step  (Scheme  1.6).  mitochondria.  C e l l u l a r G S H is c o m p a r t m e n t a l i z e d ,  residing  in the  cytosol  and  H o w e v e r , the s y n t h e s i s of G S H t a k e s p l a c e in c y t o s o l only a n d the  mitochondrial G S H is actually of c y t o p l a s m i c origin (Griffith a n d Meister, 1985).  A two  c o m p o n e n t carrier s y s t e m w a s identified for the transport of G S H into m i t o c h o n d r i a with the  high  affinity  one  being  responsible  for  the  transport  c o n c e n t r a t i o n s w e r e lower than 1 m M ( M a r t e n s s o n et a l . , 1990).  15  when  cytosolic  GSH  Methionine  Glutamate Cysteine  Glycine y-Glutamylcysteine  ATP  ADP  •+ G S H ATP  ADP  S c h e m e 1.6. Glutathione b i o s y n t h e s i s . Methionine s e r v e s a s a c y s t e i n e p r e c u r s o r in the liver ( D e L e v e a n d K a p l o w i t z , 1991).  Several  e s s e n t i a l functions  are  served  by  G S H within  the  cell  including  maintaining the r e d u c e d status of protein thiols, s c a v e n g i n g h y d r o g e n p e r o x i d e a n d organic  peroxides  when  coupled  with  GSH  peroxidase,  detoxifying  reactive  electrophiles v i a conjugation reactions, a n d providing storage for free c y s t e i n e . C e l l u l a r generation of o x y g e n radicals a n d p e r o x i d e s is a c o n t i n u o u s a n d p h y s i o l o g i c a l event upon  oxygen  consumption  (Sies and  Moss,  1978;  C h a n c e et  al.,  1979).  In  m i t o c h o n d r i a , the reactive o x y g e n s p e c i e s n e e d to be detoxified by G S H p e r o x i d a s e c o u p l e d with G S H or otherwise they would c a u s e mitochondrial dysfunction l e a d i n g to cell injury ( S i e s a n d M o s s , 1978; R e e d , 1990). O n the other h a n d , biotransformation of xenobiotic c o m p o u n d s may result in electrophilic metabolites w h i c h react with G S H ( S h a y i q et a l . , 1 9 9 1 ; S h a n et a l . , 1993).  T h i s p r o c e s s represents detoxification w h e n  the l e v e l s of reactive electrophile are low, but toxic c o n s e q u e n c e s m a y o c c u r if cellular G S H is d e p l e t e d ( R e e d , 1990). C o n j u g a t i o n of G S H with electrophiles may o c c u r s p o n t a n e o u s l y , although the reaction rates are generally e n h a n c e d by glutathione S - t r a n s f e r a s e ( G S T ) .  16  The G S T  e n z y m e s are located mainly in c y t o s o l , c o n s i s t i n g of four c l a s s e s d e s i g n a t e d a , it, \a a n d 0. (Mannervik a n d D a n i e l s o n , 1988).  In addition to the cytosolic G S T , there is a  distinct m e m b r a n e - b o u n d e n z y m e referred to a s m i c r o s o m a l G S T e v e n though it h a s b e e n found in other s u b c e l l u l a r fractions (Morgenstern et a l . , 1984; W i l c e a n d P a r k e r , 1994).  More  recently,  two  new  G S T enzymes have  been  isolated  from  the  mitochondrial matrix a n d c h a r a c t e r i z e d to s h o w that they are related to the c y t o s o l i c a a n d 0 c l a s s e s , respectively (Harris, J . M . et a l . , 1 9 9 1 ; A d d y a et a l . , 1994).  One  characteristic a s p e c t of G S T e n z y m e s is their catalytic effects on G S H conjugation with a variety  of s u b s t a n c e s including e p o x i d e s a n d a,(3-unsaturated  e s t e r s (or  a,p-  unsaturated k e t o n e s or a l d e h y d e s ) which are found in the m e t a b o l i s m of V P A (Section 1.1.3.2). T h e b a s i s of the catalytic m e c h a n i s m of G S T is that the e n z y m e ( s ) is c a p a b l e of lowering the p K  a  of the G S H thiol group. T h e p K  a  of the thiol function d r o p s from 9  in a q u e o u s solution to b e t w e e n 6 a n d 7 w h e n G S H binds to the e n z y m e proteins ( C h e n et a l . , 1 9 8 8 ; G r a m i n s k i et a l . , 1 9 8 9 a ; b).  1.1.3.2. GSH conjugation of the reactive metabolites of VPA.  It s h o u l d b e noted  that s o m e primary metabolites of V P A are subjected to further biotransformation in o r g a n e l l e s different from w h e r e the metabolites are f o r m e d .  For example, 4-ene V P A  f o r m e d in e n d o p l a s m i c reticulum c a n be taken up by mitochondria a n d m e t a b o l i z e d v i a P-oxidation to (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene V P A ) ( K a s s a h u n et a l . , 1994), a n d p o s s i b l y 2-propyl-3-oxo-4-pentenoic a c i d (3-keto-4-ene V P A ) ( K a s s a h u n et al., 1994). T h i s metabolic pathway is of toxicological interest b e c a u s e 4 - e n e V P A a n d (E)-2,4-diene V P A w e r e d e m o n s t r a t e d to be c a p a b l e of p r o d u c i n g m i c r o v e s i c u l a r  17  s t e a t o s i s in rats, a feature p h e n o m e n o n frequently s e e n in V P A hepatotoxicity (Section 1.2). T h e d i e n e metabolite c a n a l s o be formed v i a the P 4 5 0 c a t a l y z e d d e h y d r o g e n a t i o n of ( E ) - 2 - e n e V P A ( K a s s a h u n a n d Baillie, 1993). T h e conjugation of G S H with the reactive metabolites of V P A w a s d e m o n s t r a t e d in vivo in rats ( K a s s a h u n et a l . , 1 9 9 1 ; 1994).  U p o n adiministration of a d o s e of 4 - e n e  V P A , 2-propyl-5-(glutathion-S-yl)-3-pentenoic a c i d ( 5 - G S - 3 - e n e V P A ) a n d 2-propyl-5(glutathion-S-yl)-3-oxopentanoic a c i d ( 5 - G S - 3 - k e t o V P A ) w e r e d e t e c t e d a s the biliary metabolites with the former being p o s s i b l y derived from the conjugation of G S H with ( E ) - 2 , 4 - d i e n e V P A a n d the latter resulting from the G S H conjugation with 3 - k e t o - 4 - e n e V P A ( K a s s a h u n et a l . , 1 9 9 1 ; 1994). T h e metabolite 5 - G S - 3 - e n e V P A w a s d e t e c t e d in the bile of rats treated with (E)-2-ene V P A a s well ( K a s s a h u n et a l . , 1994). It h a s b e e n p r o p o s e d that the c o n j u g a t e d d o u b l e b o n d s of (E)-2,4-diene V P A are activated through the formation of the c o r r e s p o n d i n g C o A thioester either in m i t o c h o n d r i a (from 4 - e n e V P A ) or in e n d o p l a s m i c reticulum (from (E)-2-ene V P A ) to react with G S H v i a a M i c h a e l addition ( K a s s a h u n et a l . , 1994). T h e clinical r e l e v a n c e of s u c h findings r e s i d e s in the fact that 2-propyl-5-(A/-acetylcystein-S-yl)-3-pentenoic a c i d ( 5 - N A C - 3 - e n e V P A ) , the e n d product arising from the degradation of the c o r r e s p o n d i n g G S H c o n j u g a t e through the  m e r c a p t u r i c a c i d pathway, w a s detected a s a urinary  metabolite  in  patients  receiving V P A therapy ( K a s s a h u n et a l . , 1991). The  P450  catalyzed  epoxidation  of  4-ene  VPA  results  in  2-propyl-4,5-  e p o x y p e n t a n o i c a c i d (4,5-epoxy V P A ) which partitions b e t w e e n s p o n t a n e o u s hydrolysis to form 2-propyl-4,5-dihydroxy-4-pentanoic a c i d lactone (4,5-dihydroxy V P A y-lactone) (Prickett a n d Baillie, 1986) a n d conjugation with G S H . T h e resulting G S H conjugate, 2 -  18  propyl-4-hydroxy-5-(glutathion-S-yl)pentanoic  acid  lactone  (5-GS-4-hydroxy  VPA  lactone), w a s identified in the bile of rats treated with 4 - e n e V P A ( K a s s a h u n et a l . , 1994). A n o t h e r biliary G S H conjugated metabolite p o s s i b l y d e r i v e d from the reaction of G S H with 4 , 5 - e p o x y V P A w a s determined to be  2-propyl-5-hydroxy-4-(glutathion-S-  yl)pentanoic a c i d ( 4 - G S - 5 - h y d r o x y V P A ) which is a structural i s o m e r of 5 - G S - 4 - h y d r o x y V P A lactone. T h i s structural a s s i g n m e n t w a s r e g a r d e d a s tentative b e c a u s e of the lack of a synthetic reference c o m p o u n d to validate the identification ( K a s s a h u n et a l . , 1994).  1.1.4. Glucuronidation C o n j u g a t i o n with L-glucuronic acid represents the principal p h a s e II metabolic p a t h w a y of V P A a n d m a n y of its p h a s e I metabolites d u e to the p r e s e n c e of the c a r b o x y l a t e functional group. T h e excretion of V P A glucuronide typically a c c o u n t s for ~ 5 0 % of the d o s e in rats (Dickinson et a l . , 1979) a n d ~ 3 0 % in patients receiving chronic V P A therapy (Dickinson et a l . , 1989). T h e major p h a s e I a n d p h a s e II metabolic p a t h w a y s of V P A are outlined in S c h e m e s 1.7 a n d 1.8.  19  COOH  COOH p-Oxidation  Conjugation > (VPA)  ((E)-2-Ene VPA)  VPA glucuronide  P450  COOH  OH (3-Hydroxy VPA)  OH  COOH  COOH  COOH  (4-Hydroxy VPA)  (4-Ene VPA)  Scheme 1.7. Metabolism of VPA via h y d r o x y l a t i o n / d e h y d r o g e n a t i o n a n d glucuronidation.  20  p-oxidation,  OH (5-Hydroxy VPA)  P450  catalyzed  21  1.2. VPA Hepatotoxicity  1.2.1. Characteristics of VPA hepatotoxicity The  hepatotoxicity  induced  c a t e g o r i e s (Dreifuss et a l . , 1987).  by V P A h a s b e e n s u g g e s t e d to  fall  into  two  O n e is a d o s e - r e l a t e d s i d e effect w h i c h c o n s i s t s of  e l e v a t e d liver e n z y m e s a n d a p p e a r s to be reversible. S e r u m e n z y m e levels generally return to n o r m a l following a reduction of d o s a g e or discontinuation of the drug. second  hepatotoxic  independent.  category  is t e r m e d  idiosyncratic:  unpredictable  and  is  The dose  Despite its low i n c i d e n c e , idiosyncratic toxicities are reported to c a u s e a  n u m b e r of fatal hepatic injuries ( Z i m m e r m a n a n d Ishak, 1 9 8 2 ; D r e i f u s s et a l . , 1987). P a t i e n t s in the most s u s c e p t i b l e group are y o u n g children, 0-2 y e a r s o l d , w h o a r e under polytherapeutic treatment e s p e c i a l l y with phenytoin or P B in addition to V P A (Dreifuss et a l . , 1987).  During 1978 - 1984, the hepatic fatality rate for patients o n V P A  m o n o t h e r a p y under 2 y e a r s of a g e w a s 1/7000, c o m p a r e d with 1/45000 for t h o s e older than 2 y e a r s .  U n d e r polytherapy, the fatality rate i n c r e a s e d to 1/500 for the 0-2 y e a r  old group a n d 1/12000 for t h o s e older than 2 y e a r s (Dreifuss et a l . , 1987). T h e clinical s y n d r o m e  of V P A a s s o c i a t e d hepatotoxicity  includes  a n o r e x i a , n a u s e a a n d vomiting followed by fever, h y p o g l y c e m i a , a n d  lethargy,  hemorrhage  ( e v i d e n c e of p r o g r e s s i v e liver failure), a n d finally h y p e r a m m o n e m i c c o m a a n d death (Cotariu a n d Z a i d m a n , 1988). most frequent  Liver biopsy r e v e a l e d m i c r o v e s i c u l a r s t e a t o s i s a s the  lesion often a c c o m p a n i e d by centrizonal n e c r o s i s ( Z i m m e r m a n  Ishak, 1 9 8 2 ; J e a v o n s , 1984).  and  It w a s reported that liver m i c r o v e s i c u l a r s t e a t o s i s w a s  o b s e r v e d in 17 of the 21 c a s e s for w h i c h d a t a w e r e available, a n d a m o n g the 17 c a s e s ,  22  10 h a d n e c r o s i s ( Z i m m e r m a n a n d Ishak, 1982). In rats, high d o s e s of V P A w e r e s h o w n to c a u s e hepatic s t e a t o s i s (Lewis et a l . , 1 9 8 2 ; K e s t e r s o n et a l . , 1984) a n d to result in s w o l l e n or ruptured mitochondria ( J e z e q u e l , A . M . et a l . , 1984). T h e s e d a t a a p p e a r to s u g g e s t that the hepatotoxicity is a s s o c i a t e d with drug d a m a g e to m i t o c h o n d r i a , most likely the p-oxidation s y s t e m ( J e z e q u e l , A . M . et a l . , 1984). S i m i l a r c o n c l u s i o n s c a n be d r a w n from metabolic studies of V P A in h u m a n s in w h i c h the urinary excretion of dicarboxylic  acids,  indicative of  impaired fatty a c i d p-oxidation,  was  significantly  i n c r e a s e d after a d o s e of V P A ( M o r t e n s e n , 1980). T h e p-oxidation of d e c a n o i c a c i d (a m e d i u m c h a i n fatty acid) in rat liver h o m o g e n a t e (Bjorge a n d Baillie, 1985) a n d the poxidation of palmitic a c i d (a long c h a i n fatty acid) in isolated rat h e p a t o c y t e s ( C o u d e et al., 1983) w e r e found to be inhibited by V P A . T w o major h y p o t h e s e s h a v e d e v e l o p e d to rationalize the o b s e r v e d inhibition of mitochondrial p-oxidation by V P A a n d the apparent resultant hepatotoxicity.  In o n e  h y p o t h e s i s the formation of toxic metabolite(s) is s u g g e s t e d to play a key role in V P A i n d u c e d toxicity while in the other hypothesis C o A depletion is e m p h a s i z e d .  Both  h y p o t h e s e s h a v e b e e n e s t a b l i s h e d b a s e d on o b s e r v a t i o n s of V P A biotransformation.  1.2.2. Toxic metabolite hypothesis (4-ene pathway hypothesis) T h e similarity of V P A i n d u c e d hepatotoxicity to R e y e ' s s y n d r o m e a n d J a m a i c a n vomiting  s i c k n e s s led a group  of  investigators to  postulate  that 4 - e n e  V P A is  r e s p o n s i b l e for the liver injury b e c a u s e the metabolite w a s c l o s e l y related structurally to two  known  hepatotoxicants, i.e. 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 a c i d (a metabolite  of  hypoglycin) a n d 4 - p e n t e n o i c a c i d ( 4 - P A ) ( G e r b e r et a l . , 1979; Z i m m e r m a n a n d Ishak,  23  1982).  Further investigations revealed that liver m i c r o v e s i c u l a r s t e a t o s i s c o u l d be re-  produced  by  chronic  administration  of  4-ene  V P A and  (E)-2,4-diene  VPA,  an  intermediate in the p-oxidative m e t a b o l i s m of 4 - e n e V P A , to rats (100 m g / k g for 5 days) ( K e s t e r s o n et a l . , 1984).  It a p p e a r e d that s e r u m concentrations of 4 - e n e V P A w e r e  higher in pediatric patients than in older patient g r o u p s (Abbott et a l . , 1 9 8 6 a ; T a t s u h a r a et a l . , 1987), the former s u g g e s t e d to be at most risk to V P A hepatotoxicity (Dreifuss et al., 1987). W h e n tested in vitro, 4 - e n e V P A w a s cytotoxic to rat h e p a t o c y t e s ( K i n g s l e y et a l . , 1983) a n d w a s a n effective inhibitor of mitochondrial p-oxidation p r e p a r a t i o n s (Bjorge a n d Baillie, 1 9 8 5 ; P o n c h a u t et a l . , 1992).  in rat liver  R a d i o l a b e l e d 4-ene  V P A w a s s e e n to bind covalently to both rat liver proteins ( P o r u b e k et a l . , 1989) a n d P 4 5 0 e n z y m e s (Prickett a n d Baillie, 1986). Patients w h o h a d taken V P A a l o n g with the P450  enzyme  inducers,  phenytoin,  P B , or  carbamazepine  (CBZ),  were  more  s u s c e p t i b l e to V P A i n d u c e d liver injury than t h o s e under V P A m o n o t h e r a p y (4-ene V P A results from the P 4 5 0 c a t a l y z e d desaturation of V P A , S e c t i o n 1.1.2.2) ( Z i m m e r m a n a n d Ishak, 1 9 8 2 ; D r e i f u s s et a l . , 1987; C o t a r i u a n d Z a i d m a n , 1 9 8 8 ; L e v y et a l . , 1990). Based  o n the  known  fact  that  electrophile, 3-keto-4-pentenoic  p-oxidation a c i d , which  of  4 - P A produces  inhibits  3-ketoacyl  a  highly  reactive  C o A thiolase  in  a  suicidal m a n n e r ( S c h u l z , 1983), a p r o p o s e d m e c h a n i s m of 4 - e n e V P A hepatotoxicity s u g g e s t s that the biotransformation mitochondria  l e a d s to the formation  of 4 - e n e V P A (and (E)-2,4-diene V P A ) in the of 3-keto-4-ene V P A w h i c h c a n alkylate  thereby irreversibly inhibit p-oxidative e n z y m e ( s ) (Baillie, 1988).  24  and  In a c c o r d a n c e with this m e c h a n i s m , the activity of a c e t o a c e t y l - C o A t h i o l a s e w a s o b s e r v e d to be inhibited by 4 - e n e V P A in rat h e p a t o c y t e s ( P o r u b e k et a l . , 1991). H o w e v e r , d e s p i t e the detection of two p r e c u r s o r s of 3 - k e t o - 4 - e n e V P A , i.e. (E)-2,4d i e n e V P A a n d 3-hydroxy-4-ene V P A (Rettenmeier et a l . , 1985), the putative metabolite itself w a s not directly identified in either in vivo or in vitro experiments.  E v i d e n c e for a  reactive form of 4 - e n e V P A w a s obtained in this laboratory w h e n the G S H a n d Na c e t y l c y s t e i n e ( N A C ) conjugates of (E)-2,4-diene V P A w e r e d e t e c t e d in the bile a n d urine, respectively, of rats d o s e d with 4 - e n e V P A or (E)-2,4-diene V P A ( K a s s a h u n et al., 1991). species,  B a s e d on the s a m e principle that G S H m a y s e r v e a s a trap for reactive  3-keto-4-ene  V P A was subsequently  identified  a s the  GSH  conjugated  metabolite ( 5 - G S - 3 - k e t o V P A ) , albeit the amount present w a s very low ( K a s s a h u n et al., 1994).  O n the other h a n d , (E)-2,4-diene V P A w a s determined to b e the  major  metabolite of 4 - e n e V P A a n d G S H conjugation of the activated ( E ) - 2 , 4 - d i e n e V P A w a s b e l i e v e d to  occur  in  the  mitochondria  ( K a s s a h u n et  al.,  1991;  1994).  Since  mitochondria h a v e no c a t a l a s e a n d rely solely on G S H c o u p l e d with G S H p e r o x i d a s e to detoxify h y d r o p e r o x i d e s (Section 1.1.3.1),  this conjugation reaction m a y p r o d u c e a  l o c a l i z e d depletion of G S H that would result in oxidative s t r e s s with a c c o m p a n y i n g h e p a t o c e l l u l a r d a m a g e ( K a s s a h u n et a l . , 1 9 9 1 ; 1994). In support  of this  m e c h a n i s m , the  N A C conjugate  of  (E)-2,4-diene V P A ,  indicative of in vivo G S H conjugation with (E)-2,4-diene V P A , w a s d e t e c t e d in the urine of patients receiving V P A therapy with the levels being 3 - 4 times higher in t h o s e w h o h a d d e v e l o p e d V P A related liver toxicity ( K a s s a h u n et a l . , 1991). Incubation of h u m a n  25  l y m p h o c y t e s with V P A in the p r e s e n c e of m o u s e liver m i c r o s o m e s s h o w e d a V P A c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e in lymphocyte cell death a n d a significantly higher cell d e a t h o c c u r r e d in l y m p h o c y t e s from patients w h o h a d d e v e l o p e d while receiving V P A (Farrell et a l . , 1989b; Farrell a n d Abbott, 1991).  hepatotoxicity  When G S H was  a d d e d to the incubation m e d i u m , a protective effect w a s o b s e r v e d for the c e l l s towards V P A i n d u c e d cytotoxicity (Farrell et a l . , 1989b; Farrell a n d Abbott, 1991).  A small  n u m b e r of patients d e s c r i b e d a s having V P A - a s s o c i a t e d hepatotoxicity, 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 steatosis in the  livers, s u r v i v e d following  drug withdrawal  and  treatment with N A C , a precursor for G S H s y n t h e s i s (Farrell et a l . , 1989a). O n the contrary, from clinical c a s e studies of V P A - m e d i a t e d hepatotoxicity  in  w h i c h V P A metabolite profiles in patients were determined, the toxicity a p p e a r e d to be i n d e p e n d e n t of the formation of 4 - e n e V P A ( S i e m e s et a l . , 1993).  H i g h e r than normal  levels of ( E ) - 2 - e n e V P A , 2-propyl-3-pentenoic a c i d (3-ene V P A ) a n d 2-(1'-propenyl)-2p e n t e n o i c a c i d (2,3'-diene V P A ) but m u c h l e s s 3-keto V P A w e r e d e t e c t e d in the s e r u m of patients w h o h a d d e v e l o p e d apparent d r u g - i n d u c e d toxicity ( S i e m e s et a l . , 1993). S i m i l a r to that s u g g e s t e d by E a d i e et a l . ( E a d i e et a l . , 1988), t h e s e findings c o u l d be interpreted a s a n inhibition of p-oxidation reactions, p r e s u m a b l y in the c o n v e r s i o n of ( E ) - 2 - e n e V P A - C o A to 3-keto V P A - C o A , having triggered the hepatotoxicity of V P A . But S i e m e s et a l . ruled out the involvement of toxic metabolite(s) of V P A b e c a u s e of a p o o r correlation of the hepatotoxicity with either the s e r u m 4 - e n e V P A or (E)-2,4-diene V P A c o n c e n t r a t i o n s ( S i e m e s et a l . , 1993). A similar c o n c l u s i o n w a s r e a c h e d following c o m p a r a t i v e studies of V P A a n d (E)-2-ene V P A in rats w h e r e the i n c i d e n c e of liver m i c r o v e s i c u l a r s t e a t o s i s w a s o b s e r v e d to be independent of p l a s m a levels of 4 - e n e  26  V P A a n d (E)-2,4-diene V P A . It w a s therefore s u g g e s t e d that t h e s e metabolites w e r e not the d e c i s i v e factors in V P A i n d u c e d hepatotoxicity ( L o s c h e r et a l . , 1993).  1.2.3. CoA depletion hypothesis Because  C o A is a n obligate  cofactor  in the transport  of fatty a c i d s into  m i t o c h o n d r i a a n d in the s u b s e q u e n t s t e p s of fatty a c i d p-oxidation (Section 1.1.1.1), depletion of the intracellular free C o A pool d u e to the c o n v e r s i o n of  xenobiotic  carboxylic a c i d s to either inert or slowly metabolizable a c y l - C o A thioesters m a y elicit a n impairment of mitochondrial p-oxidation ( B r a s s , 1994).  In incubations of V P A with  intact rat mitochondria, V P A w a s c o n v e r t e d to the c o r r e s p o n d i n g C o A thioester (Li et al., 1991). T h i s a c y l - C o A w a s s h o w n to be poorly h y d r o l y z e d in either rat or rabbit liver preparations in c o m p a r i s o n with its structural isomer, o c t a n o y l - C o A ( M o o r e et a l . , 1988).  T h u s , a n i n c r e a s e in the acyl C o A / f r e e C o A ratio c o u l d be a s s o c i a t e d with a  depletion of the free C o A pool in the mitochondrial matrix d u e to the  competitive  participation of V P A a n d its metabolites a n d h a s thus b e e n c o n s i d e r e d a s o n e major c o u r s e for the production of V P A hepatotoxicity (Harris, R. A . et a l . , 1991). S e v e r a l lines of e v i d e n c e supporting this h y p o t h e s i s include (1) m e t a b o l i s m of V P A to 2 - e n e V P A , 3-hydroxy V P A , a n d 3-keto V P A in the h u m a n w a s d e c r e a s e d by co-administration of aspirin (acetylsalicylic acid), s u g g e s t i n g that V P A a n d aspirin w e r e involved in a competition for free C o A pools (Abbott et a l . , 1986b); (2) the r e c o v e r y of 3-keto V P A from  urinary  h u m a n subjects treated with g l u c o s e infusion  was  significantly higher than that from t h o s e in the fasting state, p o s s i b l y d u e to the fact that e n d o g e n o u s fatty acid levels are e l e v a t e d in the fasting state while t h e s e s a m e fatty  27  a c i d s are d e p r e s s e d during the glucose-infusion state ( K o c h et a l . , 1989); a n d (3) during incubation of V P A with rat liver mitochondria a m a r k e d d e c r e a s e of free C o A levels w a s a c c o m p a n i e d by a n inhibition of the oxidation of long- a n d m e d i u m - c h a i n fatty a c i d s ( B e c k e r a n d Harris, 1 9 8 3 ; Turnbull et a l . , 1983). H o w e v e r , the C o A depletion theory h a s b e e n c h a l l e n g e d by a kinetic e x p e r i m e n t in w h i c h the time c o u r s e s of C o A depletion a n d p-oxidation inhibition by V P A w e r e examined.  T h e sequestration of cellular C o A by V P A w a s o b s e r v e d to b e c o m p l e t e  within 3 0 s e c , w h e r e a s 2.5 min w a s required for V P A to i n d u c e inhibition of the poxidation of palmitoyl-carnitine ( P o n c h a u t et a l . , 1992). A l s o , similar a r g u m e n t s m a d e against the "toxic metabolite hypothesis" m a y a l s o apply here: the depletion of C o A should  be  V P A concentration  dependent,  yet  the  hepatotoxicity  a p p e a r s to  be  idiosyncratic in nature, i.e. d o s e independent.  1.3. Use of Fluorine Substituted VPA Analogues as Mechanistic Probes in VPA Hepatotoxicity  1.3.1. Site specific metabolic blockade: fluorine substitution It is well k n o w n that replacement of h y d r o g e n by fluorine in a biologically important m o l e c u l e m a y yield a n a n a l o g u e of that s u b s t a n c e with i m p r o v e d selectivity or a modified s p e c t r u m of activity ( W e l c h , 1990; P a r k a n d Kitteringham, 1994). With its s m a l l v a n der W a a l s radius (1.35 A ) , fluorine c l o s e l y r e s e m b l e s h y d r o g e n in s i z e a n d m a y mimic h y d r o g e n with respect to steric requirements for interactions with receptors a n d e n z y m e s . O n c e introduced, the high carbon-fluorine b o n d strength (112 kcal/mole)  28  renders the fluorine substitutent resistant to m a n y metabolic transformations w h i c h m a y o c c u r with c a r b o n - h y d r o g e n b o n d s (98 kcal/mol). strength  of the  carbon-fluorine  bond  a n d the  B e c a u s e of the i n c r e a s e d b o n d  e n h a n c e d lipid solubility,  fluorine-  substituted c o m p o u n d s h a v e proven to be very useful a s analytical p r o b e s  and  d i a g n o s t i c tools in biotransformation p r o c e s s e s ( W e l c h , 1990). A  good  example  was  provided  by  the  fluorination  of  paracetamol  ( a c e t a m i n o p h e n ) (Barnard et a l . , 1993). T h e P 4 5 0 c a t a l y z e d oxidation of p a r a c e t a m o l is k n o w n to p r o d u c e a reactive metabolite, n a m e l y , A / - a c e t y l - p - b e n z o q u i n o n e i m i n e ( N A P Q I ) w h i c h is normally detoxified by G S H conjugation.  W h e n a n o v e r d o s e of the  drug is g i v e n , e x t e n s i v e G S H conjugation with A/-acetyl-p-benzoquinoneimine m a y result in depletion of the hepatic G S H pool, leading to liver injury. It w a s d e m o n s t r a t e d that fluorination of p a r a c e t a m o l at the 2 a n d 6 position g a v e a c o m p o u n d that w a s less toxic than the non-fluorinated counterpart by 2-fold (Barnard et a l . , 1993). H e p a t i c G S H levels w e r e not affected upon a d o s e of the fluorinated a n a l o g u e , a s o p p o s e d to a d e c r e a s e to 5 0 % of control o b s e r v e d in a n i m a l s treated with p a r a c e t a m o l . T h u s , the r e d u c e d hepatotoxicity of 2,6-difluorinated p a r a c e t a m o l w a s b e l i e v e d to be largely d u e to the i n c r e a s e d oxidation potential of the c o m p o u n d w h i c h in turn r e d u c e d the propensity of the fluorinated a n a l o g u e s to be bioactivated through oxidative m e t a b o l i s m a n d to s e q u e s t e r hepatic G S H (Barnard et a l . , 1993).  1.3.2. Fluorine substitution at the a-position of VPA and 4-ene VPA A l t h o u g h there w a s no single report regarding fluorinated V P A a n a l o g u e s at the time of writing this P h . D . r e s e a r c h p r o p o s a l , a n u m b e r of articles h a v e s i n c e a p p e a r e d  29  a s either patents or r e s e a r c h p a p e r s p u b l i s h e d during the last three y e a r s . patent, 2-(3',3',3'-trifluoropropyl)-5,5,5-trifluoropentanoic  acid  In o n e  (5,5,5,5',5',5'-hexafluoro  V P A ) w a s s y n t h e s i z e d a n d tested a s a n anticonvulsant d r u g , a n d it w a s c l a i m e d that the  hexafluorinated  V P A a n a l o g u e h a d stronger  anticonvulsant  activity  and  less  a d v e r s e effects than V P A ( Y a m a g u c h i a n d Y a n a k a , 1992). In the present study, 2-fluoro-2-propylpentanoic a c i d (a-fluoro V P A ) a n d 2-fluoro2-propyl-4-pentenoic a c i d (a-fluoro-4-ene V P A ) w e r e p r e p a r e d a s m e c h a n i s t i c p r o b e s a n d a s potential anticonvulsant a g e n t s .  M e t a b o l i c p a t h w a y s e n v i s i o n e d for t h e s e two  fluorine-substituted V P A a n a l o g u e s are outlined in S c h e m e 1.9, a n d a r e c h a r a c t e r i z e d by retaining all the major biotransformation p a t h w a y s of V P A e x c e p t for mitochondrial |3-oxidation.  T h u s , the a-fluorinated a n a l o g u e s w o u l d be subject to P 4 5 0 c a t a l y z e d  oxidation a n d glucuronidation w h e r e a s their putative G o A thioesters w e r e not e x p e c t e d to b e s u b s t r a t e s of the p-oxidative e n z y m e s . T h e d e s i g n a n d testing of a-fluoro V P A a n a l o g u e s is b a s e d on the rationale that if the hepatotoxicity of V P A is i n d u c e d through the formation of a reactive metabolite(s), preventing the m e t a b o l i s m of 4 - e n e V P A to (E)-2,4-diene V P A v i a the p-oxidation pathway s h o u l d eliminate or m a r k e d l y r e d u c e the toxicity.  O n the other h a n d , if 4 - e n e V P A a n d a-fluoro-4-ene V P A p r o d u c e similar  levels of hepatotoxicity, then C o A depletion or metabolic p a t h w a y s other than oxidation n e e d to be taken into account.  30  p-  1.4. Thesis Objectives  T h e major a i m of this study w a s to test the s o - c a l l e d " 4 - e n e pathway" h y p o t h e s i s for V P A hepatotoxicity using a-fluorinated V P A a n a l o g u e s a s m e c h a n i s t i c p r o b e s .  In  order to m a k e a c o m p l e m e n t a r y c o m p a r i s o n b e t w e e n 4 - e n e V P A a n d its fluorinated counterpart  with respect to  m e t a b o l i s m , initial efforts f o c u s e d o n  detection  and  quantitation of G S H conjugated metabolites. S u b s e q u e n t l y , 4 - e n e V P A a n d oc-fluoro-4e n e V P A w e r e e x a m i n e d for hepatotoxicity using liver m i c r o v e s i c u l a r s t e a t o s i s a s a marker, a n d their effects on hepatic G S H status will be investigated a s contributing factors. T h e following e x p e r i m e n t s w e r e performed to r e a c h t h e s i s objectives:  (1)  T o s y n t h e s i z e a-fluoro V P A a n d a-fluoro-4-ene V P A a n d to s y n t h e s i z e  putative G S H related thiol c o n j u g a t e s which m a y derive from the m e t a b o l i s m of 4 - e n e V P A a n d a-fluoro-4-ene V P A a n d finally to s c r e e n for n e w G S H related thiol c o n j u g a t e s in treated rats using L C / M S / M S . (2)  T o isolate rat liver subcellular fractions in order to e v a l u a t e the activities of  G S T e n z y m e s in t h e s e fractions a n d to determine the role of G S T e n z y m e s in c a t a l y z i n g G S H conjugation with (E)-2,4-diene V P A . (3) T o evaluate the in vivo hepatotoxicity of 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A in rats through light m i c r o s c o p i c a n d electron m i c r o s c o p i c e x a m i n a t i o n of liver s a m p l e s ; to detect a n d quantitate (E)-2,4-diene V P A in the s e r u m a n d urine of treated rats; a n d to e x a m i n e the effects of 4 - e n e V P A a n d a-fluoro-4-ene V P A o n rat hepatic cytosolic a n d mitochondrial G S H p o o l s .  31  a-Fluoro VPA-Glucuronide  Glucuronidation  COOH  COSCoA CoA  (a-Fluoro VPA) P450 /  (a-Fluoro-5-hydroxy V P A , a-Fluoro-4-hydroxy V P A , a-Fluoro-3-hydroxy V P A )  (a-Fluoro V P A C o A thioester)  \P450  COOH  -Oxidation  COOH  (a-Fluoro-4-ene VPA)  COOH  ((E)-2,4-Diene V P A )  P450 COOH  (2-Fluoro-4,5-epoxy V P A )  GSH  o-c GS (2-Fluoro-5-GS-4hydroxy V P A lactone)  S c h e m e 1.9. M e t a b o l i c p a t h w a y s e n v i s i o n e d for a-fluorine-substituted V P A a n a l o g u e s , w h i c h a r e c h a r a c t e r i z e d by retaining all metabolic p a t h w a y s of V P A e x c e p t for (3oxidation.  32  (4)  T o establish s e r u m concentration-time profiles of 4-ene V P A a n d a-fluoro-4-  e n e V P A in rats a n d to c h a r a c t e r i z e liver uptake a n d s e r u m protein binding properties of the two c o m p o u n d s . (5) T o e v a l u a t e a-fluoro V P A a s a n anticonvulsant drug in m i c e .  33  2. Experimental  2.1. Materials  /V-Acetylcysteamine, bromide,  A/-acetyl-L-cysteine ( N A C ) ,  te/f-butyldimethylsilyl  a c r o l e i n , allyl  a l c o h o l , allyl  chloride ( t B D M S C I ) , butyllithium (1.6 M in h e x a n e s ) ,  butyric a c i d , c a l c i u m hydride, 1-chlorobutane, chlorodifluoroacetic a n h y d r i d e , 1-chloro2 , 4 - d i n i t r o b e n z e n e ( C D N B ) , chlorotrimethylsilane, (DBU),  dicyclohexylcarbodiimide  (DCC),  1,8-diazabicyclo[5.4.0]undec-7-ene digitonin,  diisopropylamine,  4-  dimethylaminopyridine ( D M A P ) , ethylbutyric a c i d , e t h y l h e x a n o i c a c i d , ethyl p e n t a n o a t e , L-glutamine,  4-heptanone,  hexamethylphosphoramide  hydroxysuccinimide, octanoic acid,  (HMPA),  1,5-pentamethylenetetrazole  a c i d ( 4 - P A ) , p o l y h y d r o g e n fluoride-pyridine  hexanoic  acid,  N-  (PTZ), 4-pentenoic  reagent, p o t a s s i u m m o n o p e r o x y sulfate  ( O X O N E ® ) , triethylamine, 2,2,2-trifluoroethanol, valeric a c i d a n d valproic a c i d ( V P A ) w e r e o b t a i n e d from A l d r i c h C h e m i c a l C o . ( M i l w a u k e e , W l ) .  T h e 4 - P A and V P A were  distilled a n d the digitonin w a s recrystallized twice from ethanol prior to u s e . A/-Fluorobenzenesulfonimide  ( N F S i ) w a s p u r c h a s e d from A l l i e d S i g n a l  Inc.  (Buffalo, N Y ) . T h e c o m m e r c i a l product N F S i w a s purified before u s e by filtration of its m e t h y l e n e chloride solution through  a short silica gel c o l u m n . T h e  reagent  was  r e c l a i m e d by evaporating the methylene chloride a n d dried at 2 5 ° C , 0.1 m m H g . Acetonitrile,  ammonium  ethylenediaminetetraacetic  acid  chloride, (EDTA),  34  di-sodium L-glycine,  hydrogen methanol,  orthophosphate, petroleum  ether,  p h e n o b a r b i t o n e s o d i u m ( P B ) , p o t a s s i u m bicarbonate, p o t a s s i u m chloride, p o t a s s i u m hydroxide, s i l i c a gel 6 0 (230 - 4 0 0 m e s h ) , s o d i u m chloride, s o d i u m  dihydrogen  o r t h o p h o s p h a t e , a n h y d r o u s s o d i u m sulfate a n d trichloroacetic a c i d w e r e o b t a i n e d from British D r u g H o u s e s , Inc. ( V a n c o u v e r , B C ) . S  p T M O c t a d e c y l extraction c o l u m n w a s from J . T. B a k e r (Phillipsburg, N J ) . e  1,4-Dithiothreitol (DTT), glutathione dinucleotide  disulfate  (GSSG),  nicotinamide  p h o s p h a t e - r e d u c e d ( N A D P H ) a n d bovine s e r u m albumin  adenine  (BSA) were  p u r c h a s e d from B o e h r i n g e r M a n n h e i m C a n a d a (Laval, P Q ) . O x y t o c i n w a s obtained from C a l b i o c h e m (La J o l l a , C A ) . Diethyl acetamide- H-|8 2  ether,  tetrahydrofuran  ([ H-|8]BSTFA) 2  (THF)  were  and  obtained  7V,A/-bis(trimethylsilyl)trifluoro-  from  Caledon  Laboratories  Ltd.  ( E d m o n t o n , A B ) . T h e T H F w a s distilled o v e r c a l c i u m hydride prior to u s e . P o l y e t h y l e n e tubing P E - 1 0 w a s obtained from C l a y A d a m s ( P a r s i p p a n y , N J ) . T h e A m i c o n micropartition s y s t e m w a s from W . R. G r a c e & C o . ( D a n v e r s , M A ) . 4-Aminobutyric  acid-2,2,3,3,4,4- H 2  ([ H ]GABA) 2  6  6  was  obtained  from  MSD  Isotopes C o . (Montreal, P Q ) . A/-Methyl-A/-(f-butyldimethylsilyl)trifluoroacetamide trimethylsilytrifluoroacetamide  (MSTFA)  and  (MTBSTFA),  A/-methyl-A/-  tris-(2-carboxyethyl)phosphine  (TCEP)  w e r e from P i e r c e C h e m i c a l C o . (Rockford, IL). L - C y s t e i n e , cysteinylglycine, 5,5'-dithio-£>/s-(nitrobenzoic hydroxyethyl)-1-piperazineethanesulfonic from Helix pomatia), glutathione  acid)  (DTNB),  a c i d ( H E P E S ) , (3-glucuronidase  ( G S H ) , glutathione  35  4-(2-  (type H-2,  r e d u c t a s e ( G R , T y p e III  from  Bakers  Y e a s t ) , glutathione  S-transferase  ( G S T , from  rat  liver),  D-mannitol,  3-  m e r c a p t o p r o p i o n i c a c i d ( 3 - M P ) , s u c r o s e , 5-sulfosalicylic a c i d , a n d trifluoroacetic a c i d ( T F A ) , Triton X - 1 0 0 , T r i z m a a n d urethane w e r e p u r c h a s e d from S i g m a C h e m i c a l C o . (St. L o u i s , M O ) . 2 - [ H 7 ] P r o p y l - 4 - p e n t e n o i c a c i d ([ H7J-4-ene V P A ) a n d (E)-2-propyl-2-pentenoic 2  2  a c i d ((E)-2-ene V P A ) w e r e s y n t h e s i z e d in this laboratory by J . J . Z h e n g a n d J . P a l a t y , respectively.  2.2. Instrumentation and Analytical Methods  2.2.1. NMR spectroscopy NMR spectrometers  spectra in  the  were  obtained  Department  of  on  Bruker  Chemistry,  WH-200  or  University  of  Bruker  WH-400  British C o l u m b i a .  C h e m i c a l shifts are e x p r e s s e d relative to tetramethylsilane.  2.2.2. High resolution MS H i g h resolution m a s s s p e c t r a were r e c o r d e d on either a K r a t o s M S 5 0 m a s s s p e c t r o m e t e r (70 e V , 1 5 0 ° C , resolution: 10000) or a K r a t o s M S 8 0 m a s s s p e c t r o m e t e r (70 e V , resolution: 7000) interfaced with a C a r l o E r b a 4 1 6 0 g a s c h r o m a t o g r a p h (DB1 c o l u m n : 15 m x 0.32 m m , 0.25 urn; helium: 30 c m / s e c ; G C : 5 0 ° C , 1 min, 5 0 - 2 5 0 ° C , 1 5 ° C / m i n ) in the Department of C h e m i s t r y , University of British C o l u m b i a .  36  2.2.3. GC/MS Qualitative G C / M S a n a l y s i s of synthetic c o m p o u n d s w a s performed o n a Hewlett P a c k a r d H P 5 7 0 0 A g a s c h r o m a t o g r a p h c o u p l e d with a M A T - 1 1 1 m a s s spectrometer. T h e G C w a s fitted with a g l a s s c o l u m n p a c k e d with 3 % D e x s i l 3 0 0 o n 100 - 2 0 0 m e s h S u p e l c o p o r t (1.8 m x 2 m m , o v e n temperature: 3 2 ° C / m i n from 5 0 to 3 0 0 ° C ) . T h e m a s s s p e c t r o m e t e r w a s o p e r a t e d u s i n g electron impact ionization (El) at a n e n e r g y of 7 0 e V a n d e m i s s i o n current of 3 0 0 m A . Qualitative G C / M S a n a l y s e s of the urinary N A C conjugate of ( E ) - 2 , 4 - d i e n e V P A a n d m o u s e brain s y n a p t o s o m a l G A B A w e r e carried out o n a Hewlett P a c k a r d 5 9 8 9 A m a s s s p e c t r o m e t e r ( M S ) c o u p l e d with a Hewlett P a c k a r d 5890II g a s c h r o m a t o g r a p h . T h e G C w a s fitted with a Hewlett P a c k a r d H P - 1 c o l u m n (12.5 m x 0.20 m m , 0.33 urn) and  helium u s e d a s the carrier g a s .  T h e G C p a r a m e t e r s for a s s a y of the N A C  conjugate w e r e : o v e n temperature: 5 0 - 2 0 0 ° C ( 3 0 ° C / m i n ) , 2 0 0 - 3 2 0 ° C ( 5 ° C / m i n ) ; a n d helium h e a d p r e s s u r e : 5 psi.  T h e G C parameters for a s s a y of G A B A w e r e : o v e n  temperature: 5 0 - 1 3 0 ° C ( 3 0 ° C / m i n ) , 1 3 0 - 2 0 0 ° C ( 1 0 ° C / m i n ) , 2 0 0 - 2 5 0 ° C  (30°C/min);  a n d helium h e a d p r e s s u r e : 3 psi (Palaty et a l . , 1994). T h e M S w a s o p e r a t e d u s i n g E l at a n e m i s s i o n current of 3 0 0 m A , a n ion s o u r c e temperature of 2 7 5 ° C a n d a n ionization e n e r g y of 7 0 e V . Qualitative a n d quantitative a n a l y s e s of brain, s e r u m a n d urinary drug a n d drug metabolites w e r e performed on a Hewlett P a c k a r d 5 9 7 1 A m a s s s e l e c t i v e detector ( M S D ) interfaced to a Hewlett P a c k a r d 5890II g a s c h r o m a t o g r a p h . G C : J & W Scientific D B - 1 7 0 1 c o l u m n (30 m x 0.25 m m , 0.25 urn); helium carrier g a s at a h e a d p r e s s u r e of  37  15 p s i ; temperature program: 8 0 - 1 0 0 ° C ( 1 0 ° C / m i n ) , 1 0 0 - 1 3 0 ° C ( 2 ° C / m i n ) , 1 3 0 - 2 6 0 ° C (30°C/min).  M S D : E l / S c a n or s e l e c t i v e ion monitoring (SIM) m o d e with a n ion s o u r c e  temperature of 1 8 0 ° C ; a n e m i s s i o n current of 3 0 0 m A a n d a n ionization e n e r g y of 7 0 e V ( Y u e t a l . , 1995).  2.2.4. LC/MS/MS L C / M S / M S experiments w e r e carried out on a F i s o n s V G Quattro t a n d e m m a s s s p e c t r o m e t e r interfaced to a Hewlett P a c k a r d 109011 liquid c h r o m a t o g r a p h .  Positive  e l e c t r o s p r a y w a s u s e d a s the m e a n s of ionization a n d c o l l i s i o n - i n d u c e d d i s s o c i a t i o n (CID) involved argon a s the target g a s at a p r e s s u r e of 3.0 x 1 0 "  4  mbar.  Other  p a r a m e t e r s w e r e capillary voltage 3.36 kV, c o n e voltage 2 9 V with s k i m m e r offset by 5 V a n d collision e n e r g y 5 0 e V . T h e multipliers 1 a n d 2 w e r e both set at 6 5 0 volts except t h o s e m e n t i o n e d e l s e w h e r e . T h e low m a s s a n d high m a s s resolutions w e r e set at 5.5 for M S 1 a n d 12.5 for M S 2 , or a s otherwise indicated.  T h e s o u r c e temperature w a s  8 0 ° C . M S / M S e x p e r i m e n t s w e r e carried out on protonated m o l e c u l a r ions. H P L C w a s performed on either a Hewlett P a c k a r d H y p e r s i l O D S c o l u m n (100 x 2.1 m m , 5 um) or a P h e n o m e n e x Ultracarb O D S 2 0 c o l u m n (150 x 1 m m , 3 }im), a n d s a m p l e s w e r e d e l i v e r e d at a flow rate of 5 0 u L / m i n .  T h e mobile p h a s e c o n s i s t e d of  methanol/water ( 0 . 0 5 % T F A ) a n d w a s p r o g r a m m e d a s follows: M e t h o d A , 7 5 % water for 2 min, a linear d e c r e a s e to 5 0 % water at 5 min, a hold at 5 0 % water to 6 min, a linear d e c r e a s e to 1 5 % water at 8 min, a hold at 1 5 % water to 3 0 min.  38  M e t h o d B, 7 5 % water for 3 min, a linear d e c r e a s e to 5 0 % water at 6 m i n , hold for 7 min, a linear d e c r e a s e to 1 5 % water at 14 min, a n d hold for 16 min. M e t h o d C , a linear d e c r e a s e of water from 7 5 % at 0 min to 5 0 % at 15 min, hold for 5 m in, a linear d e c r e a s e to 1 5 % water at 2 2 min, a n d hold for 13 min. M e t h o d D, 7 5 % water at 0 min, a linear d e c r e a s e to 5 0 % water at 5 min, hold for 7 min, a linear d e c r e a s e to 1 5 % water at 15 min, a n d hold for 15 min. In s o m e multiple reaction monitoring ( M R M ) experiments, differences in H P L C retention  times w e r e  o b s e r v e d a m o n g s e v e r a l transitions  s e l e c t e d for  a  single  c o m p o u n d in a single run. But the differences w e r e generally l e s s than 0.2 m i n .  2.2.5. HPLC H P L C purification of c o m p o u n d s of interest w a s performed on a Hewlett P a c k a r d 1050 liquid c h r o m a t o g r a p h fitted with either a W h a t m a n Partisil O D S 2 c o l u m n (250 x 9 mm, 5 urn) or a Hewlett P a c k a r d S p h e r i s o r b O D S 2 c o l u m n (250 x 4 m m , 5 u.m) u s i n g U V detection at 2 1 0 n m . T h e mobile p h a s e c o n s i s t e d of acetonitrile/water ( 0 . 0 5 % T F A , v/v) a s detailed in the following s e c t i o n s . T h e H P L C c a p a c i t y factors of V P A , 4 - e n e V P A , a-fluoro V P A a n d a - f l u o r o - 4 - e n e V P A w e r e d e t e r m i n e d o n a Hewlett P a c k a r d 1050 liquid c h r o m a t o g r a p h fitted with a Hewlett P a c k a r d S p h e r i s o r b O D S 2 c o l u m n (250 x 4 m m , 5 |im) u s i n g U V detection at 2 1 0 n m . T h e mobile p h a s e c o n s i s t e d of acetonitrile/phosphate buffer (50 m M , p H 2.5, 4 5 / 5 5 , v/v) at a flow rate of 1.0 m L / m i n .  39  2.2.6. Spectrophotometry Spectrophotometric m e a s u r e m e n t s w e r e c o n d u c t e d on a Hewlett P a c k a r d 8 4 5 2 A D i o d e A r r a y spectrophotometer.  2.2.7. Polarography P o l a r o g r a p h i c m e a s u r e m e n t s of oxidative phosphorylation w e r e performed on a G i l s o n K-IC o x y g r a p h in the Department of Biochemistry, University of British C o l u m b i a . T h e electrode w a s fitted with a s t a n d a r d Y S I m e m b r a n e ( Y e l l o w s p r i n g s , O H ) .  2.3. Chemical Synthesis  2.3.1. Synthesis of 2-propyl-4-pentenoic acid (4-ene VPA) T h e title c o m p o u n d w a s s y n t h e s i z e d v i a the alkylation of ethyl p e n t a n o a t e with allyl bromide in the p r e s e n c e of lithium d i i s o p r o p y l a m i d e ( L D A ) followed by alkaline hydrolysis ( K a s s a h u n et a l . , 1991). (63-64°C/0.02 mmHg).  T h e product w a s purified by fractional distillation  G C / M S m a s s s p e c t r u m ( t B D M S derivative): m/z  (%) 199 (100,  M+-57), 7 5 (100), 129 (10), 157 (5). H N M R ( C D C I ) : 5 0.85 (t, 3 H , J H = 7 H z , C H ) , 1  3  1.20-1.40  (m,  2H,  CH CH ), 2  3  1.40-1.65  (m,  2H,  H  CH CH ), 2  2  3  2.10-2.35  (m,  2H,  C H C H = C H ) , 2 . 3 5 - 2 . 5 0 (m, 1 H , C H C O O H ) , 4.98 (d, 1 H , J H = 10 H z , C H = C H ) , 5.03 2  H  2  2  (d, 1 H , J H = 17 H z , C H = C H ) , 5.70 (tdd, 1 H , J H = 7, 10, 17 H z , C H = C H ) , 11.22 (s, H  H  2  1H.COOH).  40  2  2.3.2. Synthesis of (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene VPA) T h e title c o m p o u n d w a s p r e p a r e d through the alkylation of ethyl p e n t a n o a t e with a c r o l e i n in the p r e s e n c e of L D A , followed by hydro-mesyloxy-elimination a n d alkaline h y d r o l y s i s ( K a s s a h u n et a l . , 1991). T h e product w a s purified by flash c h r o m a t o g r a p h y (silica gel 6 0 , 2 3 0 - 4 0 0 m e s h ) : 2 0 % ether in h e x a n e (v/v) followed by 1 0 0 % ether. G C / M S m a s s s p e c t r u m ( t B D M S derivative): m/z (%) 197 (100, M (17), 9 5 (17), 123 (10), 2 3 9 (3, M+-15), 2 5 4 (trace, M+). J  H  = 7 H z , C H ) , 1.4 (sextet, 2 H , J  H  3  = C C H ) , 5,42 (d, 1 H , J 2  H  H  H  H  1  +  - 57), 7 5 (47), 155  H N M R ( C D C I ) : 5 0.9 (t, 3 H , 3  = 7 H z , C H C H ) , 2.3 (t, 2 H , J 2  3  H  H  = 7 Hz,  = 11 H z , C H = C H ) , 5.6 (d, 1 H , J H = 17 H z , C H = C H ) , 6.5H  2  2  6.7 (m, 1 H , C H = C H ) , 7.22 (d, 1 H , J H = 11 H z , O C C = C H ) . (E) : (Z) = 9 2 : 8 b a s e d on H  2  a G C / M S assay.  2.3.3. Synthesis of Ethyl (E)-2-propyl-2-pentenoate ((E)-2-ene VPA ethyl ester) ( E ) - 2 - E n e V P A ethyl ester w a s s y n t h e s i z e d through the alkylation of  ethyl  p e n t a n o a t e with p r o p i o n a l d e h y d e in the p r e s e n c e of L D A , followed by h y d r o - m e s y l o x y elimination ( L e e , 1991). G C / M S m a s s s p e c t r u m : m/z 6 7 (30), 135 (30), 141 (28), 170 (M+, 28).  (%) 5 5 (100), 113 (62), 9 5 (45),  1H N M R ( C D C I 3 ) : 5 0.88 (t, 3 H , J H H  = 7 Hz,  C H C H ) , 1.03 (t, 3 H , J|—11—| = 7 H z , = C H C H C H ) , 1.25 (t, 3 H , J H = 7 H z , O C H C H ) , 2  3  2  H  3  2  3  1.40 (sextet, 2 H , J H = 7 H z , C H C H ) , 2.18 (pentad, 2 H , J H = 7 H z , C = C H C H ) , H  2.25 (t, 2 H , J  H  H  2  H  3  = 7 H z , = C C H ) , 4.16 (q, 2 H , J 2  Hz, C=CH).  41  H  H  2  = 7 H z , O C H ) , 6.72 (t, 1 H , J 2  H  H  = 7  2.3.4. Synthesis of 2,2-difluoro-4-pentenoic acid (F2-4-PA) T h e F 2 - 4 - P A w a s s y n t h e s i z e d with modifications to a p r o c e d u r e d e s c r i b e d by G r e u t e r et a l . (Greuter et a l . , 1988). T o allyl a l c o h o l (0.1 m o l , 6.0 g) a n d triethylamine (0.1 m o l , 10.1 g) in ether w a s a d d e d chlorodifluoroacetic a n h y d r i d e (0.1 m o l , 2 5 g) at 0°C.  T h e mixture w a s stirred at room temperature for a n additional 2 h before  partitioning b e t w e e n ether a n d water.  Allyl chlorodifluoroacetate w a s o b t a i n e d upon  r e m o v a l of ether a n d distilled at 100 - 1 0 3 ° C , yield: 9 0 % . T o allyl chlorodifluoroacetate (75 m m o l , 10 g) in acetonitrile (44 ml_) w a s a d d e d d r o p w i s e chlorotrimethylsilane (88 m m o l , 9.5 g) a n d stirred at room temperature for 1 h. F r e s h l y activated z i n c dust (4.5 g) w a s a d d e d a n d the resultant mixture w a s h e a t e d to reflux at 1 0 0 ° C for 2 0 h.  Acetonitrile w a s rotor-evaporated u n d e r v a c u u m from the  mixture to o n e half of the original v o l u m e a n d the residue w a s mixed with s i l i c a g e l (40 g) in water (45 ml_), a n d stirred at room temperature for 12 h. S i l i c a g e l w a s r e m o v e d by filtration a n d the acetonitrile r e m o v e d by rotor-evaporation under v a c c u m .  The  r e s i d u e w a s partitioned b e t w e e n water a n d ether, a n d c r u d e product w a s o b t a i n e d upon removal of the ether layer by e v a p o r a t i o n . T h e d e s i r e d c o m p o u n d w a s o b t a i n e d by fractional distillation ( 5 8 - 5 9 ° C / 5 m m H g , yield: 8 0 % ) . G C / M S m a s s s p e c t r u m ( T M S derivative): m/z{%) 77 (100), 7 3 (30), 115 (17), 1 9 3 (17) (M+-15).  1  H N M R (CDCI3):  2.86 (2t, 2 H , J|—11—| = 7 H z , Jp|_| = 16 H z , C H ) , 5.30 (d, 1 H , J H = 10 H z ; d , 1 H , J H  2  H z ; C H = C H ) , 5 . 7 5 (tdd, 1 H , J 2  13C N M R  (CDCI3): 6 3 8 . 8 9  122.31 (s, C - 5 ) , 126.38 (t, J  (t, J C  F  m  8  = 17  H  H  = 7, 10, 18 H z , C H = C H ) , 1 0 . 2 5 (s, 1 H , C O O H ) .  C  F  = 2 3 . 5 H z , C - 3 ) , 114.98 (t, J  2  = 5.42 H z , C - 4 ) , 168.71 (t, J  42  C  F  C  F  = 249.5 Hz, C-2),  = 33.3 Hz, C-1).  2.3.5. Synthesis of 2-propyl-2-fluoro-pentanoic acid (a-fluoro VPA) 2.3.5.1. Procedure A:  T o d i i s o p r o p y l a m i n e (43 m m o l , 6 ml_) in T H F (50 ml_) w a s  a d d e d butyllithium (43 m m o l , 2 7 ml_) at - 7 8 ° C .  After stirring at - 1 0 ° C for 3 0 min, the  mixture w a s c o o l e d to - 7 8 ° C , H M P A (43 m m o l , 7.5 ml_) in T H F (10 mL) w a s a d d e d , a n d the resultant mixture w a s stirred for 15 min.  T o this L D A / T H F / H M P A solution w a s  a d d e d ethyl 2-propylpentanoate (30 m m o l , 4 g) in T H F (7 mL). T h e mixture w a s stirred for 6 0 min before N F S i (49 m m o l , 16 g) in T H F (50 mL) w a s a d d e d . T h e reaction w a s kept at - 7 8 ° C for 120 min, a l l o w e d to reach room temperature overnight, a n d q u e n c h e d by a d d i n g saturated a q u e o u s a m m o n i u m chloride. T h e mixture w a s acidified to p H 1 with 6 M hydrochloric a c i d solution a n d then extracted with diethyl ether twice. combined  organic  layer w a s w a s h e d c o n s e c u t i v e l y with a q u e o u s  The  hydroxylamine  hydrochloride solution, saturated s o d i u m bicarbonate solution a n d water, then rotore v a p o r a t e d u n d e r v a c u u m to give ethyl 2-fluoro-2-propylpentanoate. product w a s purified by fractional distillation ( 2 9 - 3 0 ° C / 0 . 0 2 m m H g ) . spectrum:  m/z(%)  found. H N M R 1  (CDCI ):  3  crude  G C / M S mass  29 (47), 5 5 (100), 9 7 (42), 117 (18), 148 (33). N o m o l e c u l a r ion w a s 3  5 0.78 (t, 6 H ,  J  H  H  = 7 H z , 2 x C H 3 ) , 1.10 (t, 3 H ,  O C H C H ) , 1.00-1.80 (m, 8 H , 2 x C H C H ) , 4.05 (q, 2 H , J 2  The  2  2  M  J H H  = 7 Hz,  = 7 Hz, O C H ) . 2  a - F l u o r o V P A w a s obtained upon alkaline hydrolysis of the ethyl e s t e r a n d w a s purified by fractional distillation ( 5 6 - 5 7 ° C / 0 . 0 5 m m H g , y i e l d : 8 0 % ) . m a s s s p e c t r u m of the t B D M S derivative (M - tBu)+: m/z ( F S i ( C H ) ) + : m/z 3  2  H i g h resolution  219.1212, calc. 219.1217;  7 7 . 0 2 2 7 , c a l c . 7 7 . 0 2 2 3 ; a n d the [ H ] T M S derivative (M - C D ) + : 2  9  43  3  m/z  2 2 5 . 1 6 0 7 , c a l c . 2 2 5 . 1 5 8 7 ; ( F S i ( C D ) ) : m/z  83.0583, calc. 83.0593.  +  3  2  H NMR  1  ( C D C I ) : 8 0.89 (t, 6 H , J H = 7 H Z , 2 X C H ) , 1.20-1.60 (m, 4 H , 2 x C H C H ) , 1.78-2.05 H  3  (m, 4 H , C H C H ) . 3c 2  4'), 3 9 . 2 8 (d, J  C  F  2  N M R ( C D C I 3 ) : 5 13.95 (s, C - 5 , 5'), 16.52 (d, J  1  2  3  = 22.1 H z , C - 3 , 3'), 9 7 . 5 5 (d, J  C  F  3  C  F  = 3.5 H z , C - 4 ,  = 185.7 H z , C - 2 ) , 177.72 (d, J  C  F  =  26.8 H z , C-1).  2.3.5.2. Procedure B:  T o a n a q u e o u s solution (50 ml_) of s o d i u m c y a n i d e (0.5 m o l ,  2 5 g) w a s a d d e d a m m o n i u m chloride (30 g) in water (60 ml_), followed by c o n c e n t r a t e d a m m o n i a solution (43.5 m L ) . T h e mixture w a s c o o l e d in a ice-water bath a n d a solution of 4 - h e p t a n o n e (0.5 mol, 5 7 g) in methanol (80 mL) w a s a d d e d d r o p w i s e .  T h e flask  w a s then s t o p p e r e d a n d half i m m e r s e d for 5 h in a water bath at 5 5 - 6 0 ° C .  Upon  c o o l i n g the a q u e o u s layer w a s extracted three times with diethyl ether. T h e c o m b i n e d organic  layer  was  evaporated  aminodipropylacetonitrile.  under  vacuum  H  H  afford  an  oily  product  of  2-  G C / M S m a s s s p e c t r u m : m / z ( % ) 4 3 (100), 5 7 ( 1 0 0 ) , 70(100),  98(38), 28(27), 85(25), no m o l e c u l a r ion at m/z J  to  140.  1  H N M R ( C D C L ) : 8 0.82 (t, 6 H , 3  = 7 H z , 2 x C H ) , 1.2-1.5 (m, 8 H , 2 x C H C H ) , 1.6 (s, N H ) . 3  2  2-Aminodipropylacetonitrile  2  obtained  above  2  was  dissolved  in  concentrated  hydrochloric a c i d solution at 0 ° C a n d refluxed for 2 4 h. T h e mixture w a s overnight  at  room temperature.  collected  by  filtration.  evaporation  of  the  T h e crystallized a m i n o  Several crops aqueous  of  precipitate  solution.  Free  were  stirred  a c i d hydrochloride obtained  upon  2-aminodipropylacetic  was  further  acid  recrystallized in ethanol after neutralizing the hydrochloride salt with diethylamine.  was 1  H  N M R ( D 0 ) : 8 0.78 (t, 6 H , J = 7 H z , 2 x C H ) , 0.90-1.32 (m, 4 H , 2 x C H C H ) , 1.402  3  1.70 (m, 4 H , 2 x H C C ( N H ) C O O H ) . 2  2  44  2  3  T o 2 - a m i n o d i p r o p y l a c e t i c a c i d (20 m m o l , 3.18 g) a n d s o d i u m fluoride (30 m m o l , 1.26 g) in 15 m L of p o l y h y d r o g e n fluoride-pyridine reagent w a s a d d e d dry pyridine (2 m L ) d r o p w i s e at 0 ° C under nitrogen, followed by the addition in portions of s o d i u m nitrate (2.24 g) at 0 ° C . T h e mixture w a s stirred at 0 ° C for 1 h a n d at r o o m temperature for additional a n 4 h before it w a s partitioned b e t w e e n i c e - c o l d water (50 m L ) a n d diethyl ether (50 m L ) . T h e water layer w a s further extracted with diethyl ether twice. T h e c o m b i n e d ether layer w a s w a s h e d with saturated s o d i u m chloride solution twice (30  m L e a c h ) a n d i c e - c o l d water  (30 m L ) , dried o v e r m a g n e s i u m sulfate, a n d  e v a p o r a t e d to afford c r u d e product.  2.3.6. Synthesis of 2-propyl-2-fluoro-4-pentenoic acid (oc-fluoro-4-ene VPA) Ethyl 2-fluoro-2-propyl-4-pentenoate w a s s y n t h e s i z e d from ethyl  2-propyl-4-  p e n t e n o a t e following a similar p r o c e d u r e a s d e s c r i b e d a b o v e (procedure A for a-fluoro V P A ) . T h e c r u d e product w a s purified by flash c h r o m a t o g r a p y (silica g e l 6 0 , 2 3 0 - 4 0 0 m e s h , diethyl ether/petroleum ether = 10/90, v/v, y i e l d : 8 6 % ) . G C / M S m a s s s p e c t r u m :  m/z(%)  9 5 (100), 139 (65), 41 (65), 7 3 (57), 5 5 (44), 1 4 6 (44), 111 (39), 1 6 8 (26), 1 5 7  (11), 1 8 8 (M+, 2).  1  H N M R ( C D C I ) : 6 0.78 (t, 3 H , J H = 7 H z , C H ) , 1.07 (t, 3 H , J H = H  3  H  3  7 H z , O C H C H ) , 1.15-1.45 (m, 2 H , C H C H ) , 1.50-1.80 (m, 2 H , C F C H C H ) , 2 . 6 0 2  3  (m, 2 H , J H = 7 H z , J H  (dd, J  H  H  2  F  H  3  2  2  = 2 5 H z , C H C H = C H ) , 4 . 1 0 (q, 2 H , J H = 7 H z , O C H ) , 4 . 9 2 2  H  2  2  = 15, 11 H z , C H = C H ) , 5.45-5.73 (m, 1 H , C H = C H ) . 2  2  a - F l u o r o - 4 - e n e V P A w a s obtained upon alkaline h y d r o l y s i s of the ethyl e s t e r a n d w a s purified by fractional distillation ( 5 6 - 5 7 ° C / 0 . 0 5 m m H g , y i e l d : 8 0 % ) . G C / M S m a s s  45  s p e c t r u m ( t B D M S derivative): m / z ( % ) 7 7 (100), 2 1 7 (48, M+-57), 7 3 (47), 9 5 (33), 133 (10), 189 (3).  1  H N M R ( C D C I ) : 6 0.93 (t, 3 H , J H = 7 H z , C H ) , 1.20-1.65 (m, 2 H , J H H  3  H  3  = 7 H z , C H C H ) , 1.75-2.00 (m, 2 H , C F C H C H ) , 2.61 (dd, 2 H , J H = 7 H z , J 2  3  2  H  2  F  H  = 20,  2 4 H z , C H C H = C H ) , 5.17 (dd, 2 H , J|_JH = 11, 1 6 H z , C H = C H ) , 5.80 (tdd, 1 H , J H = 7, 2  2  H  2  10, 16 H z , C H = C H ) . 1 3 C N M R ( C D C I 3 ) : 5 13.92 (s, C - 5 ' ) , 16.51 (d, J 2  4'), 3 8 . 7 2 (d, J  C  F  = 2 1 . 8 H z , C - 3 ' ) , 4 1 . 4 3 (d, J  H z , C - 2 ) , 119.99 (s, C - 5 ) , 130.31 (d, J  C  F  C  F  C  F  = 3.5 H z , C -  = 2 1 . 8 H z , C - 3 ) , 9 6 . 9 3 (d, J  = 3.8 H z , C - 4 ) , 1 7 7 . 2 3 (d, J  C  F  C  F  = 187.6  = 26.5 Hz, C -  1)-  2.3.7.  Synthesis of (E)-2-fluoro-2-propyl-3-pentenoic acid ((E)-a-fluoro-3-ene  VPA) Ethyl  (E)-2-fluoro-2-propyl-3-pentenoate  w a s s y n t h e s i z e d from  ethyl  (E)-2-  propyl-2-pentenoate following a similar p r o c e d u r e a s d e s c r i b e d a b o v e (procedure A for a-fluoro V P A ) . T h e c r u d e product w a s purified by fractional distillation ( 3 4 - 3 5 ° C / 0 . 0 5 m m H g ) . G C / M S m a s s s p e c t r u m : m/z (%) 7 3 (100), 115 (62), 5 5 (25), 9 5 (20), 4 3 (17), 1H N M R ( C D C I 3 ) : 5 0.91 (t, 3 H , J H = 7 H z ,  102 (15), 168 (7), 140 (5), 188 (M+, 2).  H  C H C H ) , 1.30 (t, 3 H , J H = 7 H z , O C H C H ) , 1.73 (d, 3 H , J H = 7 H z , C H = C H C H ) , 2  H  3  2  H  3  3  4 . 2 3 (q, 2 H , J H = 7 H z , O C H ) , 5.58 (dd, 1 H , J H = 17 H z , J H  H  2  F  H  = 18 H z ,  C H = C H C H ) , 5.88 (qd, 1 H , J H = 7 H z , J H = 16 H z , C H = C H C H ) . 3  H  H  3  ( E ) - a - F l u o r o - 3 - e n e V P A w a s obtained upon b a s e hydrolysis of the ethyl ester (yield : 9 0 % ) . G C / M S m a s s s p e c t r u m ( t B D M S derivative): m/z (%) 7 7 (100), 2 1 7 (51, M+-57), 115 (27), 147 (14).  1H N M R ( C D C I ) : 5 0 . 9 3 (t, 3 H , J H =7 H z , C H C H ) , 3  46  H  2  3  1.15-1.60 (m, 2 H , C H C H ) , 1.73 (d, 3 H , J H =7 H z , C H = C H C H ) , 1.75-2.10 (m, 2 H , 2  C F C H ) , 5.58 (dd, 1 H , J 2  H  3  H  H  = 16 H z , J  H z , J H = 16 H z , C H = C H C H ) . H  3  13c  F  C  F  = 18.0 H z , C F C H = C H ) , 5.90 (qd, 1 H , J H = 7  H  H  N M R ( C D C I ) : 8 13.89 (s, C - 3 ' ) , 1 6 . 3 9 (d, J 3  3.0 H z , C - 2 ' ) , 17.65 (s, C - 5 ) , 39.51 (d, J C - 2 ) , 1 2 7 . 5 0 (d, J  3  C  F  = 22.2 H z , C - 1 ' ) , 9 5 . 5 7 (d, J  = 2 0 . 3 H z , C - 3 ) , 128.20 (d, J  C  F  C  F  C  =  F  = 186.5 H z ,  = 10.7 H z , C - 4 ) , 1 7 5 . 5 0 (d, J  C  =  F  27.1 H z , C - 1 ) . ( E ) - a - F l u o r o - 3 - e n e V P A converts to 2-propyl-2-pentenoic a c i d - 4 - l a c t o n e (2-ene4 - h y d r o x y - V P A lactone) s p o n t a n e o u s l y at room temperature.  m/z(%)  G C / M S m a s s spectrum:  41 (100), 9 7 (82), 6 9 (65), 140 (M+, 38), 78 (23), 5 5 (22), 111 (22), 125 (17).  1H N M R ( C D C I ) : 8 0.75 (t, 3 H , J = 7 H z , C H C H ) , 1.14 (d, 3 H , J = 7 H z , O C H C H ) , 3  2  3  3  1.31 (sextet, 2 H , J = 7 H z , C H C H ) , 1.98 (t, 2 H , J = 7 H z , C H C H C H ) , 4 . 7 5 (qd, 1 H , 2  3  2  J = 7 H z , 2 H z , = C H C H C H ) , 6.79 (d, 1 H , J = 2 H z , = C H C H ) . 3  2  3  1 C N M R (CDCI ): 8 3  3  13.56 ( 1 C , C - 3 ' ) , 19.09 ( 1 C , C - 2 ' ) , 20.61 ( 1 C , C - 5 ) , 2 7 . 0 6 ( 1 C , C - 1 ' ) , 7 7 . 4 8 ( 1 C , C - 4 ) , 1 3 3 . 7 9 ( 1 C , C - 3 ) , 149.36 ( 1 C , C - 2 ) , 173.99 ( 1 C , C = 0 ) .  2.3.8. Synthesis of 2-propyl-2-hydroxy-pentanoic acid (a-hydroxy VPA) T o a n a q u e o u s solution (37.5 mL) of s o d i u m c y a n i d e (0.5 m o l , 2 4 . 5 g) w a s a d d e d d r o p w i s e 4 - h e p t a n o n e (0.5 mol, 5 7 g) a n d acetic a c i d (0.45 m o l , 2 7 g) in diethyl ether (100 mL) with v i g o r o u s stirring at 10 - 2 0 ° C . temperature overnight.  T h e mixture w a s stirred at room  A c e t i c a c i d (0.1 mol, 6 g) w a s a d d e d d r o p w i s e a n d the stirring  w a s c o n t i n u e d for a n additional 1 5 - 2 0 min. After the o r g a n i c layer w a s s e p a r a t e d , the aqueous  phase  was  extracted  with  diethyl  47  ether  twice.  Crude  2-hydroxy-  dipropylacetonitrile w a s obtained upon removal of solvent  in vacuo  in the p r e s e n c e of  c h l o r o a c e t i c a c i d (0.5 g) a n d further purified by distillation at 7 8 - 7 9 ° C / 0 . 0 2 m m H g . G C / M S m a s s spectrum: 141.  1  m/z{%)  71 (100), 114 (62), 99 (8), 8 6 (8).  N o m o l e c u l a r ion at  H N M R ( C D C I ) : 8 1.0 (t, 6 H , 2 x C H ) , 1 . 4 - 1 . 8 (m, 8 H , 4 x C H ) , 3.3 (s, 1 H , 3  3  2  OH). a-Hydroxydipropylacetonitrile  (0.5  g)  obtained  above  was  dissolved  in  c o n c e n t r a t e d hydrochloric a c i d (5 mL) a n d methanol (3 mL). T h e mixture w a s refluxed for 4 8 h a n d then e v a p o r a t e d to d r y n e s s  in vacuo.  T h e r e s i d u e w a s w a s h e d with ethyl  a c e t a t e , the precipitate filtered off a n d the solvent e v a p o r a t e d to d r y n e s s  in vacuo.  The  r e s i d u e w a s further d i s s o l v e d in chloroform, filtered a n d dried with a n h y d r o u s s o d i u m sulfate.  a - H y d r o x y V P A w a s obtained upon removal of the solvent.  s p e c t r u m ( T M S b/s-derivative): 1  m/z{%)  G C / M S mass  187 (100), 147 (51), 261 (44), 2 8 9 (M+ - 15, 9).  H N M R (CDCI3): 6 0.9 (t, 6 H , 2 x C H ) , 1 . 1 - 1 . 3 (m, 2 H , C H ) , 1 . 4 - 1 . 8 (m, 6 H , 3 x 3  2  CH ). 2  2.3.9. Synthesis of A^-(2-fluoro-2-propyl-4-pentenoyl)glutamine (oc-fluoro-4-ene VPA-GIn) T o oc-fluoro-4-ene V P A (2.5 m m o l , 0.40 g) in 7.5 m L of dry d i o x a n e at 1 0 ° C w a s a d d e d /V-hydroxysuccinimide (3 m m o l , 0.35 g) followed by D C C (3 m m o l , 0.62 g).  The  mixture w a s stirred at room temperature for 5 h. T h e precipitate ( D C U ) w a s filtered off. T h e filtrate w a s a d d e d to a n a q u e o u s solution of L-glutamine (3.8 m m o l , 0.55 g) a n d s o d i u m b i c a r b o n a t e (3.8 m m o l , 0.32g), a n d stirred at 5 0 - 6 0 ° C for 2 4 h.  48  Dioxane was  e v a p o r a t e d in vacuo a s m u c h a s p o s s i b l e . T h e residue w a s w a s h e d with ethyl a c e t a t e twice, acidified to p H 2 , a n d extracted with diethyl ether three times. T h e c r u d e product o b t a i n e d u p o n r e m o v a l of ether w a s precipitated in m e t h y l e n e chloride.  LC/MS/MS  m a s s s p e c t r u m : m/z (%) 2 8 9 (MH+, 1 0 0 ) , 1 3 0 (29), 9 5 ( 1 0 ) , 1 6 0 ( 6 ) , 1 1 5 ( 6 ) , 1 2 3 (5). 1  H N M R ( C D O D ) : 8 0 . 9 5 (2t, 3 H , J 3  (dt,  1H, J  H  H  =  4,  8  Hz, NHCH),  H = 7 H z , C H ) , 1 . 2 0 - 2 . 7 5 (m, 1 0 H , 5 x C H ) , 4 . 4 0  H  3  5.08-5.22  2  (m, 2 H , C H = C H ) , 2  5.70-6.00  (m, 1 H ,  CH=CH ). 2  2.3.10. Synthesis of /v^-^-fluoro^-propylpentanoyOglutamine (a-fluoro VPA-GIn) T h e glutamine conjugate of a-fluoro V P A w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e . L C / M S / M S m a s s s p e c t r u m : m/z(%) 2 9 1 (MH+, 1 0 0 ) , 1 3 0 (30), 9 7 ( 1 2 ) , 1 6 2 (6). 1  H N M R ( C D 3 O D ) : 8 0 . 9 0 (2t, 6 H , J  4 . 4 0 (dt, 1 H , J|—11—| = 5 , 9  H  H = 7 H Z , 2 x C H 3 ) , 1 . 1 5 - 2 . 5 0 (m, 1 2 H , 6 x C H ) , 2  Hz, NHCH).  2.3.11. Synthesis of W-acetyl-S-((E)-2-propyl-2,4-pentadienoyl)cysteamine (2,4diene VPA-NACA) T o ( E ) - 2 , 4 - d i e n e V P A (5.7 m m o l , 0 . 8 g) in dry m e t h y l e n e c h l o r i d e (5 m L ) w a s a d d e d D M A P ( 0 . 6 4 m m o l , 7 8 mg) a n d A/-acetylcysteamine (5.9 m m o l , 0 . 7 g). D C C (7.1 m m o l , 1.5 g) w a s a d d e d at 0 ° C . T h e mixture w a s stirred at room t e m p e r a t u r e for 4 8 h f o l l o w e d by w a s h i n g c o n s e c u t i v e l y with a q u e o u s hydrochloric a c i d solution ( 1 . 0 M ) , s a t u r a t e d s o d i u m b i c a r b o n a t e solution a n d water.  C r u d e product w a s purified by flash  c h r o m a t o g r a p h y (silica g e l 6 0 , 2 3 0 - 4 0 0 m e s h ) : 3 0 % ether in h e x a n e f o l l o w e d by 8 0 %  49  G C / M S m a s s s p e c t r u m : m/z (%) 1 2 3 (100), 9 5 (57), 6 7 (45), 8 6  ether in h e x a n e (v/v).  (33), 2 1 2 (trace, M+-29), 241 (trace, M+). T h e isomeric ratio (E)/(Z) w a s 9 4 / 6 b a s e d o n peak areas (GC/MS). (sextet, 2 H , J  H  H  1  H N M R ( C D C I ) : 8 0.9 (t, 3 H , J 3  H  = 7 Hz, C H C H ) , 2  1.9 (s, 3 H , C O C H ) , 2.4 (t, 2 H , J  = 7 Hz, C H C H ) , 2  H  3  3  H  3  H  1.4  = 7 Hz,  = C C H ) , 3.1 (t, 2 H , J H = 7 H z , S C H ) , 3.4 (q, 2 H , J H = 7 H z , C H N H ) , 5.4 (d, 1 H , H  2  JHH =  1  1  H z  H  2  - C H = C H ) , 5.6 (d, 1 H , J 2  H  2  = 17 H z , C H = C H ) , 6.5-6.7 (m, 1 H , C H = C H ) ,  H  2  2  7.1 (d, 1 H , J H = 11 H z , O C C = C H ) . H  2.3.12.  Synthesis  of  A/-acetyl-S-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-  cysteamine (5-GS-3-ene VPA-NACA) T o 2 , 4 - d i e n e V P A - N A C A (0.41 mmol, 100 mg) in acetonitrile (4 mL) w a s a d d e d G S H (0.16 m m o l , 5 0 mg) in p h o s p h a t e buffer (0.1 M , 4 m L , p H 7.8). T h e mixture w a s stirred at room temperature for 5 h, acidified to p H 6.5 a n d w a s h e d with ether. v o l u m e of the a q u e o u s p h a s e w a s r e d u c e d purified  by H P L C :  Whatman  in vacuo  Partisil O D S 2  to 2 0 0 u l a n d c r u d e product  c o l u m n , 2.5 m L / m i n  acetonitrile/water (19/81, v/v, 0 . 0 5 % T F A , v/v) mobile p h a s e . eluted at 10.1 m i n . L C / M S / M S m a s s s p e c t r u m :  The  m/z(%)  flow  rate  and  The compound was  5 4 9 ( M H + , 100), 1 2 3 (57), 2 2 6  (17), 4 2 0 (17), 4 7 4 (12). 1H N M R ( D 0 ) : 8 0.7 (t, 3 H , J H = 7 H z , C H C H ) , 0 . 9 5 - 1 . 2 5 H  2  2  3  (m, C H C H ) , 1.25-1.60 (m, 2 H , C H C H ) , 1.75 (s, 3 H , N A C A C O C H ) , 2 . 0 5 (q, 2 H , 2  3  2  3  J H = 7 H z , G l u , C H C H ) , 2.4 (t, 2 H , J H = 7 H z , G l u C H C O ) , 2 . 5 0 - 2 . 7 5 (m, 2 H , C y s H  2  H  2  C H ) , 2 . 8 5 (t, 2 H , J H = 6 H z , N A C A C H ) , 3.0 (d, 2 H , J H = 6 H z , S C H C H = ) , 3 . 1 2 2  H  H  2  50  2  3.25 (m, 3 H , C H C O , N A C A C H ) , 3.82 (s, 2 H , G l y C H ) , 3 . 9 0 (t, H, J 2  C H ) , 4 . 3 5 (dd, 1 H , J  2.3.13.  H  2  H  H  = 6 Hz, Glu  = 6 H z , C y s C H ) , 5.4-5.5 (m, 2 H , C H = C H ) .  H  Synthesis of 2-propyl-5-(glutathion-S-yl)-3-pentenoic acid (5-GS-3-ene  VPA) T o (E)-2,4-diene V P A (5.7 m m o l , 0.8 g) in dry methylene c h l o r i d e (5 mL) w a s a d d e d 4-dimethylaminopyridine m m o l , 0.59 g).  (0.64 m m o l , 7 8 mg) a n d 2,2,2-trifluoroethanol  (5.9  D i c y c l o h e x y l c a r b o d i i m i d e (7.1 m m o l , 1.5 g) w a s a d d e d at 0 ° C . T h e  mixture w a s stirred at room temperature for 2 4 h followed by w a s h i n g c o n s e c u t i v e l y with a q u e o u s hydrochloric a c i d solution (1.0 M), saturated s o d i u m b i c a r b o n a t e solution and  water.  Trifluoroethyl  (E)-2-propyl-2,4-pentadienoate  was obtained  through  distillation at 6 8 ° C / 2 . 5 m m H g . G C / M S m a s s s p e c t r u m : m/z (%) 9 5 (100), 2 2 2 (M+, 42), 123 (32), 165 (17), 193 (12), 179 (8), 2 0 7 (5). T o a n a q u e o u s solution of G S H (0.16 mmol or 5 0 m g / 5 m L , p H 10.5) w a s a d d e d trifluoroethyl (E)-2-propyl-2,4-pentadienoate (0.45 m m o l , 100 mg) in acetonitrile (2 m L ) . T h e mixture w a s stirred at room temperature for 2 4 h, acidified to p H 2.5 a n d w a s h e d with ethyl acetate. T h e v o l u m e of the a q u e o u s p h a s e w a s r e d u c e d and  crude  product  acetonitrile/water, spectrum:  purified  by  HPLC:  Whatman  in vacuo  Partisil  ODS2  1 5 / 8 5 ; flow rate, 2.5 m L / m i n ; tR, 10.5 min.  m/z(%)  to 2 0 0 | i L column;  LC/MS/MS  mass  4 4 8 (MH+, 100), 162 (51), 2 1 6 (31), 3 1 9 (21), 2 5 6 (18), 3 7 3 (9).  1  N M R ( D 0 ) : 8 0.9 (t, 3 H , J H = 7 H z , C H C H ) , 1.1-1.4 (m, C H C H ) , 1.4-1.7 (m, 2 H , H  2  2  3  2  3  C H C H ) , 2.2 (q, 2 H , J H = 7 H z , G l u , C H C H ) , 2.5 (t, 2 H , J H = 7 H z , G l u C H C O ) , 2  H  2  51  H  2  H  2.7-2.9 (m, 2 H , C y s C H ) , 3.0-3.2 (m, 3 H , S C H C H = , C H C O ) , 3.9 (t, H , J H = 6 H z , 2  H  2  G l u C H ) , 4 . 0 (s, 2 H , G l y C H ) , 4 . 5 (dd, 1 H , J 2  H  H  = 6 H z , C y s C H ) , 5.5-5.7 (m, 2 H ,  CH=CH).  2.3.14. Synthesis of 2-propyl-5-(glycine-cystein-S-yl)-3-pentenoic acid (5-cysgly3-ene VPA) The  title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e .  HPLC  purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 1 8 / 8 2 ; flow rate, 2.5 m L / m i n ; t , 11.5 min. L C / M S / M S m a s s s p e c t r u m : m/z (%)  3 1 9 ( M H + , 100), 162 (18),  R  141 (10), 1 2 3 (4), 2 1 6 (4), 2 5 6 (3).  1  H N M R ( D 0 ) : 6 0.9 (t, 3 H , J H = 7 H z , C H C H ) , H  2  2  3  1.1-1.4 (m, 2 H , C H C H ) , 1.4-1.7 (m, 2 H , C H C H ) , 2.9-3.1 (m, 2 H , C y s C H ) , 3.1-3.3 2  3  2  2  (m, 3 H , S C H C H = , C H C O ) , 4.0 (2s, 2 H , G l y C H ) , 4 . 5 (dd, 1 H , J H = 6 H z , C y s C H ) , 2  H  2  5.5-5.7 (m, 2 H , C H = C H ) .  2.3.15. Synthesis of 2-propyl-5-(cystein-S-yl)-3-pentenoic acid (5-cys-3-ene VPA) The  title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e .  HPLC  purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 1 5 / 8 5 ; flow rate, 2.5 m L / m i n ; t , 9.0 min. L C / M S / M S m a s s s p e c t r u m : m/z (%) R  123 (4).  1  2 6 2 ( M H + , 100), 141 (11),  H N M R ( D 0 ) : 8 0.9 (t, 3 H , J H = 7 H z , C H C H ) , 1.1-1.4 (m, 2 H , C H C H ) , H  2  2  3  2  3  1.4-1.7 (m, 2 H , C H C H ) , 2.9-3.1 (m, 2 H , C y s C H ) , 3.1-3.3 (m, 3 H , S C H C H = , C H C O ) , 2  2  4.2 (dd, 1 H , J H = 6 H z , C y s C H ) , 5.5-5.7 (m, 2 H , C H = C H ) . H  52  2  2.3.16. Synthesis of 2-propyl-5-(/V-acetylcystein-S-yl)-3-pentenoic acid (5-NAC-3ene VPA) T h e title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e e x c e p t that 5NAC-3-ene  V P A w a s r e c o v e r e d from the ethyl acetate extracts.  HPLC  purification:  W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 2 2 / 7 8 ; flow rate, 2.5 m L / m i n ; tR, 10 min.  L C / M S / M S m a s s s p e c t r u m : m/z (%)  3 0 4 (MH+, 100), 123(31), 130 (20), 164 (6),  141 (6), 146 (6), 2 5 8 (5), 162 (4), 2 1 6 (3). CH CH ), 2  3  1.1-1.4 (m, 2 H , C H C H ) , 2  3  1  H N M R ( D 0 ) : 5 0.8 (t, 3 H , J H = 7 H z , H  2  1.4-1.7 (m, 2 H , C H C H ) , 2  1.9  (s, 3 H , N A C  C O C H ) , 2.7-2.9 (m, 2 H , N A C C H ) , 2.9-3.1 (m, 3 H , S C H C H = , C H C O ) , 4 . 5 (dd, 1 H , 3  2  2  J H = 6 H z , N A C N H C H ) , 5.4-5.6 (m, 2 H , C H = C H ) . H  2.3.17.  Synthesis  cysteamine (5-GS-2-ene  of  A/-acetyl-S-(2-propyl-5-(glutathion-S-yl)-2-pentenoyl)-  VPA-NACA)  T o a n a q u e o u s mixture (80 m L , 5 % of e t h a n o l , v/v) of 2 , 4 - d i e n e  VPA-NACA  (0.41 m m o l , 100 mg) a n d G S H (0.16 m m o l , 5 0 mg) at p H 6.5 w a s a d d e d rat liver cytosolic fraction (0.6 m L , for its preparation, s e e S e c t i o n 2.7). T h e reaction p r o c e e d e d at 2 5 ° C for 120 min a n d w a s q u e n c h e d by a d d i n g 1 0 % a q u e o u s T F A (v/v, 5 0 u,L). T h e mixture w a s w a s h e d with ethyl acetate, the v o l u m e of the a q u e o u s p h a s e r e d u c e d in  vacuo  to 2 0 0 u l a n d the precipitate r e m o v e d by centrifugation at 1 3 , 6 0 0 g for 15 min.  H P L C purification: Hewlett P a c k a r d S p h e r i s o r b O D S 2 c o l u m n ; acetonitrile/water, 1 9 / 8 1 ; flow rate, 1.0 m L / m i n ; t , 9.0 min. L C / M S / M S m a s s s p e c t r u m : m/z (%) 5 4 9 (MH+, 100), R  123 (89), 2 2 6 (34), 4 2 0 (5), 4 7 5 (5).  1  H N M R ( D 0 ) : 5 0.7 (t, 3 H , J H 2  53  H  = 7 Hz,  C H C H ) , 1.2 (sextet, 2 H , J H = 7 H z , C H C H ) , 1.75 (s, 3 H , N A C A C O C H ) , 2.002  H  3  2  3  3  2.20 (m, 4 H , C H C H = , G l u C H C O ) , 2.30-2.45 (m, 4 H , = C C H , G l u , C H C H ) , 2 . 5 5 2  2  2  2  2 . 9 5 (m, 6 H , S C H C H , N A C A C H , C y s C H ) , 3.2 (t, 2 H , J H = 6 H z , N A C A C H ) , 2  2  2  3.82 (s, 2 H , G l y C H ) , 3.85 (t, 1 H , 2  J H H  H  2  = 6 H z , G l u C H ) , 4.4 (dd, 1 H ,  2  J H H  = 6 Hz, Cys  C H ) , 6.6 (t, 1 H , J H = 7 H z , C H = C ) . H  2.3.18. Synthesis of 2-propyl-5-(glutathion-S-yl)-2-pentenoic acid (5-GS-2-ene VPA) T h e a q u e o u s solution of 5 - G S - 2 - e n e V P A - N A C A w a s adjusted to p H 10.5 with a q u e o u s s o d i u m hydroxide solution (3 M) a n d stirred at room temperature for 4 8 h. T h e mixture w a s acidified to p H 2.5 with 1 0 % a q u e o u s T F A (v/v), w a s h e d with ethyl  in vacuo  acetate and evaporated s p e c t r u m : m/z  to d r y n e s s to afford the product.  L C / M S / M S mass  (%) 4 4 8 (MH+, 100), 123 (64), 2 2 6 (25), 3 7 3 (8), 3 1 9 (7).  1H N M R  ( D 0 ) : 5 0.9 (t, 3 H , J H = 7 H z , C H C H ) , 1.5 (sextet, 2 H , J H = 7 H z , C H C H ) , 2 . 1 H  2  2  H  3  2  3  2.3 (m, 4 H , C H C H = , G l u C H C O ) , 2.5-2.7 (m, 4 H , = C C H , G l u , C H C H ) , 2.7-2.9 (m, 2  2  2  2  2 H , S C H C H ) , 2.9-3.1 (m, 2 H , C y s C H ) , 3.9 (t, 1 H , J H = 6 H z , G l u C H ) , 4.0 (s, 2 H , 2  2  G l y C H ) , 4.6 (dd, 1 H , J 2  H  2  H  H  = 6 H z , C y s C H ) , 6.8 (t, 1 H , J H = 7 H z , C H = C ) . H  2.3.19. Synthesis of 2-propyl-5-(glutathion-S-yl)-4-hydroxypentanoic acid lactone (5-GS-4-hydroxy VPA lactone) T o s o d i u m bicarbonate (34 m m o l , 2.88 g), a c e t o n e (12 mL) a n d water (6 mL) in a distillation a p p a r a t u s with the receiver i m m e r s e d in dry i c e - a c e t o n e bath w a s a d d e d  54  p o t a s s i u m m o n o p e r o x y sulfate (10 m m o l , 6 g).  T h e reaction mixture w a s stirred  v i g o r o u s l y at room temperature a n d a v a c u u m (water aspirator) a p p l i e d . T h e effluent w a s c o l l e c t e d a s a yellow solution c o n s i s t i n g of dimethyldioxirane a n d a c e t o n e (Curci e t a l . , 1980). T o the solution of dimethyldioxirane p r e p a r e d a b o v e w a s a d d e d ethyl 2-propyl-4p e n t e n o a t e (0.6 m m o l , 100 mg). T h e mixture w a s stirred at room temperature for 18 h a n d ethyl 2 - p r o p y l - 4 , 5 - e p o x y p e n t a n o a t e obtained upon removal of solvent G C / M S m a s s s p e c t r u m : m/z (%)  in vacuo.  101 (100), 130 (42), 141 (38), 115 (22), 157 (5), 186  (M+, 2). T o a n a q u e o u s solution G S H (0.31 mmol or 5 0 mg/5 m L , p H 9.5) w a s a d d e d ethyl 2 - p r o p y l - 4 , 5 - e p o x y p e n t a n o a t e in acetonitrile (2 mL).  T h e mixture w a s stirred at  room temperature for 2 4 h, w a s h e d with diethyl ether, acidified to p H 2.5 a n d extracted T h e v o l u m e of the a q u e o u s p h a s e w a s r e d u c e d in  with ethyl a c e t a t e three times.  vacuo to  2 0 0 u L a n d c r u d e product purified by H P L C : W h a t m a n Partisil O D S 2 c o l u m n ;  acetonitrile/water, 13/87; flow rate, 2.5 m L / m i n ; tR, 10 min. L C / M S / M S m a s s s p e c t r u m : m/z  (%)  4 4 8 (MH+, 100), 2 1 6 (39), 3 1 9 (38), 3 7 3 (19), 2 8 4 (15).  1  H NMR (D 0): 5 2  0.85 (t, 3 H , J H = 7 H z , C H C H ) , 1.2-1.8 (m, 4 H , C H C H ) , 2.1-2.3 (m, 3 H , C O C H , H  2  3  2  2  G l u , C H C H ) , 2.5 (t, 2 H , J H = 7 H z , G l u C H C O ) , 2.8-3.2 (m, 6 H , C y s C H , S C H , 2  H  2  C H C H C O ) , 3.9 (s, 2 H , G l y C H ) , 4.1 (t, 1 H , J 2  2  H z , C y s C H ) , 4.9 (m, 1 H , O C H ) .  55  H  2  H  = 6 H z , G l u C H ) , 4 . 5 (m, 1 H , J  2  H  H  = 6  2.3.20.  Synthesis of 2-propyl-5-(glycine-cystein-S-yl)-4-hydroxypentanoic acid  lactone (5-cysgly-4-hydroxy VPA lactone) The  title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e .  HPLC  purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 16/84; flow rate, 2.5 m L / m i n ; t , 10.7 m i n . L C / M S / M S m a s s s p e c t r u m : m/z (%) 3 1 9 ( M H + , 100), 2 1 6 (8), R  177 (8), 2 8 4 (4), 141 (5). (m, 4 H , C H C H ) , 2  2  1  H N M R ( D 0 ) : 5 0.9 (t, 3 H , J H = 7 H z , C H C H ) ,  2.1-2.3  H  2  (m, 1 H , C O C H ) ,  2.5-3.2  2  1.2-1.8  3  (m, 6 H , C y s C H , S C H , 2  2  C H C H C O ) , 4 . 0 (s, 2 H , G l y C H ) , 4 . 3 (m, 1 H , J H = 6 H z , C y s C H ) , 4.9 (m, 1 H , O C H ) . 2  H  2  2.3.21. Synthesis of 2-propyl-5-(cystein-S-yl)-4-hydroxypentanoic acid lactone (5cys-4-hydroxy VPA lactone) The  title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e .  HPLC  purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 1 3 / 8 7 ; flow rate, 2.5 m L / m i n ; t , 8.9 min. L C / M S / M S m a s s s p e c t r u m : m/z (%) 2 6 2 (MH+, 100), 2 4 5 (6), 120 R  (3).  1  H N M R ( D 0 ) : 5 0.9 (t, 3 H , J H = 7 H z , C H C H ) , 1.2-1.8 (m, 4 H , C H C H ) , 2 . 1 2  H  2  3  2  2  2.3 (m, 1 H , C O C H ) , 2.5-3.2 (m, 6 H , C y s C H , S C H , C H C H C O ) , 4 . 3 (m, 1 H , J H = 6 2  2  2  H  H z , C y s C H ) , 4.9 (m, 1 H , O C H ) .  2.3.22. Synthesis of 2-propyl-5-(W-acetylcystein-S-yl)-4-hydroxypentanoic acid lactone (5-NAC-4-hydroxy VPA lactone) T h e title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e e x c e p t that 5N A C - 4 - h y d r o x y V P A lactone w a s r e c o v e r e d from the ethyl a c e t a t e extracts.  56  HPLC  purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 1 5 / 8 5 ; flow rate, 2.5 m L / m i n ; t , 10.2 min. L C / M S / M S m a s s s p e c t r u m : m/z (%) R  130 (14), 2 1 6 (11), 162 (8), 2 4 5 (6), 2 5 8 (6). CH CH ), 2  3  1.2-1.8 (m, 4 H , C H C H ) , 2  2  1  3 0 4 (MH+, 100), 2 6 2 (14),  H N M R ( D 0 ) : 5 0.85 (t, 3 H , J H = 7 H z , H  2  2.05 (s, 3 H , N A C C O C H ) ,  2 . 1 - 2 . 3 (m, 1 H ,  3  C O C H ) , 2.5-3.2 (m, 6 H , N A C C H , S C H , C H C H C O ) , 4.6 (m, 1 H , J H = 6 H z , N A C 2  2  H  2  N H C H ) , 4.9 (m, 1 H , O C H ) .  Synthesis of 2-propyl-2-fluoro-5-(glutathion-S-yl)-4-hydroxypentanoic acid  2.3.23.  lactone (5-GS-2-fluoro-4-hydroxy VPA lactone) T o the solution of dimethyldioxirane p r e p a r e d a b o v e (Section 2.3.19) w a s a d d e d ethyl 2-propyl-2-fluoro-4-pentenoate (0.6 m m o l , 100 mg).  T h e mixture w a s stirred at  room temperature for 18 h a n d ethyl 2-propyl-2-fluoro-4,5-epoxypentanoate u p o n r e m o v a l of solvent  in vacuo.  G C / M S mass spectrum:  m/z  obtained  (%) 91 (100), 131 (93),  148 (93), 184 (14), 2 0 4 (M+, 14). T o a n a q u e o u s solution of G S H (0.31 mmol or 5 0 mg/5 m L , p H 9.5) w a s a d d e d ethyl 2-propyl-2-fluoro-4,5-epoxypentanoate in acetonitrile (2 m L ) .  T h e mixture w a s  stirred at room temperature for 2 4 h, w a s h e d with diethyl ether, acidified to p H 2.5 a n d extracted with ethyl acetate three times. reduced  in vacuo  T h e v o l u m e of the a q u e o u s p h a s e w a s  to 2 0 0 u l a n d c r u d e product purified by H P L C : W h a t m a n Partisil  O D S 2 c o l u m n ; acetonitrile/water, 13/87; flow rate, 2.5 m L / m i n ; t , 10 min. R  mass spectrum: 1  m/z(%)  LC/MS/MS  4 6 6 (MH+, 100), 3 3 7 (28), 2 3 4 (26), 391 (9), 3 2 0 (9), 177 (9).  H N M R ( D 0 ) : 5 0.9 (t, 3 H , J H = 7 H z , C H C H ) , 1.3-1.5 (m, 2 H , C H C H ) , 1.7-2.1 2  H  2  57  3  2  3  (m, 2 H , C F C H ) , 2.2 (q, 2 H , J H = 7 H z , G l u C H C H ) , 2.6 (t, 2 H , J H = 7 H z , G l u H  2  H  2  C H C O ) , 2.8-3.2 (m, 6 H , C y s C H , S C H , C H C F C O ) , 3.9 (s, 2 H , G l y C H ) , 4 . 0 (t, 1 H , 2  2  2  2  2  = 6 H z , G l u C H ) , 4 . 5 5 (m, 1 H , C y s C H ) , 4.9 (m, 1 H , O C H ) .  J H H  2.3.24.  Synthesis  of  2-propyl-2-fluoro-5-(/V-acetylcystein-S-yl)-4-hydroxy-  pentanoic acid lactone (5-NAC-2-fluoro-4-hydroxy VPA lactone) T h e title c o m p o u n d w a s s y n t h e s i z e d similarly a s d e s c r i b e d a b o v e e x c e p t that 5N A C - 2 - f l u o r o - 4 - h y d r o x y V P A lactone w a s r e c o v e r e d from the ethyl a c e t a t e extracts. H P L C purification: W h a t m a n Partisil O D S 2 c o l u m n ; acetonitrile/water, 1 5 / 8 5 ; flow rate, 2.5 m L / m i n ; t , 10 min. L C / M S / M S m a s s s p e c t r u m : R  162 (15), 130 (11), 2 6 3 (9), 2 3 4 (8).  1  m/z(%)  3 2 2 (MH+, 100), 2 8 0 (16),  H N M R ( D 0 ) : 6 0.85 (t, 3 H , J H = 7 H z , H  2  C H C H ) , 1.2-1.9 (m, 4 H , C H C H ) , 1.9 (s, N A C COCH3), 2.2-3.2 (m, 6 H , N A C C H , 2  3  2  2  2  S C H , C H C F ) , 4 . 5 5 (m, 1 H , N A C N H C H ) , 4.9 (m, 1 H , O C H ) . 2  2  2.3.25.  Biosynthesis of 1-0-(2-Propyl-2,4-pentadienoyl)-p-D-glucuronide (2,4-  diene VPA-glucuronide) R a t s w e r e d o s e d with (E)-2,4-diene V P A a n d the bile w a s c o l l e c t e d for 6 h a s detailed  in the  following  section  (Section 2.6).  Purification  of  2,4-diene V P A -  g l u c u r o n i d e w a s c a r r i e d out with modifications to the p r o c e d u r e d e s c r i b e d for isolation of V P A - g l u c u r o n i d e (Williams et a l . , 1992).  Briefly, bile (4 mL) c o l l e c t e d from rats  treated with (E)-2,4-diene V P A w a s acidified to p H 2, w a s h e d with 1-chlorobutane (3 x 12 mL) a n d further extracted by diethyl ether ( 3 x 1 2 mL). T h e c o m b i n e d ether extracts  58  w e r e e v a p o r a t e d to d r y n e s s under nitrogen. T h e residue w a s d i s s o l v e d in a minimum amount of methanol a n d subjected to purification by flash c h r o m a t o g r a p h y (silica gel 6 0 , 2 3 0 - 4 0 0 m e s h ) . T h e c o l u m n h a d a s i z e of 120 x 2.5 m m , a n d the eluting s o l v e n t s c o n s i s t e d initially of ether (100%) followed by ether/methanol (60/30, v/v). m a s s s p e c t r u m , m/z  (%):  141 (100), 3 1 7 (76, MH+), 123 (10), 159 (7).  LC/MS/MS 1  H NMR  ( C D O D ) : 6 0.9 (t, 3 H , J|_11_| = 7 H z , C H ) , 1.5 (sextet, 2 H , J H = 7 H z , C H C H ) , 2.3 (t, 3  H  3  2  3  2 H , J H = 7 H z , = C C H ) , 3.3-3.8 (m, 4 H , glucuronic a c i d 4 x C H ) , 5,4-5.8 (m, 3 H , H  2  C H = C H ; g l u c u r o n i c a c i d C H ) , 6.7-6.9 (m, 1 H , C H = C H ) , 7.4 (d, 1 H , J H H = 2  1  2  1  H Z  -  OCC=CH).  2.4. Determination of the lipophilicity and the ionization constants of VPA, 4-ene VPA, a-fluoro VPA and a-fluoro-4-ene VPA  T h e octanol-water partition coefficients (P) of V P A , 4 - e n e V P A , a-fluoro V P A a n d a - f l u o r o - 4 - e n e V P A w e r e determined from their H P L C c a p a c i t y factors (Abbott a n d A c h e a m p o n g , 1988). Briefly, a s t a n d a r d c u r v e w h i c h correlated H P L C c a p a c i t y factors of s t a n d a r d c o m p o u n d s (butyric a c i d , 0.998; valeric a c i d , 1.721; ethylbutyric  acid,  2 . 4 3 8 ; h e x a n o i c a c i d , 2 . 9 1 9 ; e t h y l h e x a n o i c a c i d , 6.958) with their literature log P v a l u e s (0.98, 1.51, 1.68, 1.93, 2.64) ( K e a n e et a l . , 1983) w a s constructed with a correlation coefficient r  2  > 0.99 a n d u s e d to calculate the log P v a l u e s of V P A , 4 - e n e V P A , a-fluoro  V P A a n d a-fluoro-4-ene V P A .  59  T h e apparent p K  a  v a l u e s of V P A , 4 - e n e V P A , a-fluoro V P A a n d a-fluoro-4-ene  V P A w e r e d e t e r m i n e d by potentiometric titration.  T h e c o m p o u n d s d i s s o l v e d in a 1 0 %  a q u e o u s methanol solution (2 m M , 5 0 mL) w e r e titrated with a s t a n d a r d i z e d p o t a s s i u m hydroxide solution (10 m M ) at room temperature (Abbott a n d A c h e a m p o n g , 1988).  2.5. Detection and Characterization of Drug-Associated Thiol Conjugates in Rats Treated with 4-Ene VPA or (E)-2,4-Diene VPA  2.5.1. Animal experiments M a l e S p r a g u e - D a w l e y rats ( V a n c o u v e r , B C ) w e i g h i n g 2 3 0 to 2 8 0 g w e r e a l l o w e d free a c c e s s to food ( P u r i n a Laboratory C h o w , PMI Inc,) a n d water. T h e y w e r e h o u s e d in regular c a g e s a n d e x p o s e d to a controlled 12 h c y c l e of light a n d d a r k n e s s prior to the metabolic study. A group of three rats w e r e a n e s t h e t i z e d with urethane (1 g/kg) a n d their bile ducts c a n n u l a t e d with P E - 1 0 tubing.  Control bile w a s c o l l e c t e d for 15 min.  An  a q u e o u s solution (pH 7) of 4 - e n e V P A or (E)-2,4-diene V P A w a s then a d m i n i s t e r e d at 0.7 m m o l / k g by i.p. injection a n d bile collected for a n additional 6 h. A  second  group  of  three  rats,  after  control  urine  was  collected,  were  a d m i n i s t e r e d a n a q u e o u s solution of (E)-2,4-ene V P A (pH 7) at 0.7 m m o l / k g by i.p. injection a n d h o u s e d in metabolic c a g e s to collect urine for 2 4 h. O n e rat w a s treated the s a m e a s a b o v e but d o s e d with [ H 7 ] - 4 - e n e V P A in a n 2  a q u e o u s solution (pH 7) at 0.7 m m o l / k g .  -  60  2.5.2.  Isolation of the GSH-glucuronide di-conjugates of (E)-2,4-diene VPA from  the bile of rats treated with (E)-2,4-diene VPA T h e bile (4 mL) c o l l e c t e d from rats administered (E)-2,4-diene V P A w a s acidified to p H 2, w a s h e d c o n s e c u t i v e l y with 1-chlorobutane ( 3 x 1 2 mL) a n d diethyl ether (3 x 12 mL) a n d then e v a p o r a t e d  in vacuo to  d r y n e s s . T h e r e s i d u e w a s d i s s o l v e d in water  (200 (iL) a n d subjected to purification by H P L C : Hewlett P a c k a r d S p h e r i s o r b O D S 2 c o l u m n , acetonitrile/water, 13/87; flow rate, 1.0 m L / m i n . T w o fractions w e r e c o l l e c t e d . H P L C fraction 1 ( t  R  = 7.7 min):  2-0-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-(3-  D-glucuronide ( 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II).  L C / M S / M S m a s s s p e c t r u m , m/z  6 2 4 (MH+, 100), 2 9 9 (17), 123 (14), 141 (9), 162 (9), 4 9 5 (8), 5 4 9 (5).  1  (%):  H NMR (D 0), 2  5 0.85 (t, 3 H , J H = 7 H z , C H C H ) , 1.2-1.35 (m, C H C H ) , 1.5-1.8 (2m, 2 H , C H C H ) , H  2  3  2  3  2  2.2 (m, 2 H , G l u , C H C H ) , 2.55 (m, 2 H , G l u C H C O ) , 2 . 7 - 2 . 9 5 (2m, 2 H , C y s C H ) , 3 . 1 2  2  2  3.3 (m, 3 H , S C H C H = , C H C O ) , 3.45-3.65 (m, 3 H , glucuronic a c i d 3 x C H ) , 3.9 (m, 3 H , 2  G l u C H , G l y C H ) , 4.1 (d, 1 H , glucuronic a c i d C H ) , 4.5 (dd, 1 H , J H = 6 H z , C y s C H ) , H  2  5.5-5.7 (m, 3 H , glucuronic a c i d C H ; C H = C H ) . 1 -0-(2-Propyl-5-(glutathion-S-yl)-2-pentenoyl)-p-D-glucuronide V P A - g l u c u r o n i d e ) (present a s the impurity  in H P L C fraction  1).  (5-GS-2-ene L C / M S / M S mass  s p e c t r u m , m/z (%): 6 2 4 (MH+, 100), 4 4 8 (28), 2 2 6 (13), 123 (12), 3 1 9 (8), 3 7 3 (5). N M R ( D 0 ) , 5 1.4 (sextet, 2 H , J H 2  H  = 7 H z , C H C H ) , 2.3 (t, 2 H , J H 2  3  H  1  H  = 7 Hz,  S C H C H ) , 6.96 (t, 1 H , J H H = 7 H z , C = C H ) . O t h e r s i g n a l s are likely s u p e r i m p o s e d by 2  2  t h o s e of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II.  61  H P L C fraction 2 (tR = 9.9 min):  1-0-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-p-  D-glucuronide ( 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I).  L C / M S / M S m a s s s p e c t r u m , m/z  6 2 4 (MH+, 100), 4 4 8 (32), 3 1 9 (20), 162 (14), 2 1 6 (11), 2 5 6 (6). (t, 3 H , J  H  H  1  (%)  H N M R ( D 0 ) : 8 0.85 2  = 7 H z , C H C H ) , 1.2-1.35 (m, C H C H ) , 1.5-1.8 (2m, 2 H , C H C H ) , 2.2 (m, 2  3  2  3  2  2 H , G l u , C H C H ) , 2.55 (m, 2 H , G l u C H C O ) , 2.7-2.95 (2m, 2 H , C y s C H ) , 3.1-3.3 (m, 2  2  2  3 H , S C H C H = , C H C O ) , 3.45-3.65 (m, 3 H , glucuronic a c i d 3 x C H ) , 3.9 (m, 3 H , G l u C H , 2  G l y C H ) , 4.1 (d, 1 H , glucuronic a c i d C H ) , 4.5 (dd, 1 H , J 2  H  H  = 6 H z , C y s C H ) , 5.5-5.7 (d  + m, 3 H , glucuronic a c i d C H ; C H = C H ) .  2.5.3. Detection of the GSH conjugates of (E)-2,4-diene VPA in the bile of rats treated with (E)-2,4-diene VPA A n aliquot of rat bile w a s mixed with a n equivalent v o l u m e of 0 . 0 5 % a q u e o u s T F A (v/v) a n d the precipitate r e m o v e d v i a centrifugation at 13,600a; for 15 min.  The  s a m p l e (2 u l ) w a s injected onto the Hewlett P a c k a r d H y p e r s i l O D S c o l u m n a n d eluted u s i n g H P L C m e t h o d B (Section 2.2.4).  M S / M S in M R M m o d e (transitions m/z 4 4 8 - »  123 a n d 4 4 8 -> 2 2 6 for 5 - G S - 2 - e n e V P A a n d transitions m/z 4 4 8 -> 162 a n d 4 4 8 -> 2 5 6 for 5 - G S - 3 - e n e V P A ) w a s u s e d for s e l e c t i v e detection of the metabolites. dwell t i m e s w e r e set at 2 s e c .  62  The  2.5.4. Detection of the biliary GSH-glucuronide, and the biliary and urinary NACglucuronide di-conjugates of (E)-2,4-diene VPA in (E)-2,4-diene VPA treated rats Rat bile (100 uL) w a s mixed with a n equivalent v o l u m e of a q u e o u s T F A (0.05%) a n d t h e precipitate r e m o v e d v i a centrifugation at 13,600a; for 15 min. R a t urine (500 \i L) w a s acidified to p H 3 a n d a p p l i e d to a C-JS extraction cartridge w h i c h w a s p r e w a s h e d with m e t h a n o l a n d water. and methanol.  T h e c o l u m n w a s c o n s e c u t i v e l y w a s h e d with water  T h e methanol eluate w a s e v a p o r a t e d  in vacuo  to d r y n e s s a n d the  r e s i d u e re-constituted in a q u e o u s T F A (0.05%, 1 0 0 u L ) . A n aliquot of t h e bile or urine s a m p l e s (2 - 2 0 fiL) w a s injected onto the Hewlett P a c k a r d H y p e r s i l O D S c o l u m n a n d eluted using H P L C m e t h o d A . T o record full daughter ion s p e c t r a of the metabolites, M S / M S dwell times w e r e adjusted to provide a s c a n rate of ~ 1 s e c / 1 0 0 a m u . W h e n t h e M S / M S w a s o p e r a t e d in M R M m o d e , three transitions, m/z 4 8 0 - » 3 0 4 , 4 8 0 - » 2 8 6 a n d 4 8 0 - » 1 2 3 w e r e u s e d a s criteria for the detection of t h e urinary N A C - g l u c u r o n i d e di-conjugate.  2.5.5.  Quantitation of the biliary GSH conjugate and the GSH-glucuronide di-  conjugate in (E)-2,4-Diene VPA treated rats A n aliquot of bile s a m p l e (100 |iL) w a s mixed with a n e q u i v a l e n t v o l u m e of aqueous  T F A (0.05%)  containing  5 - G S - 2 - f l u o r o - 4 - h y d r o x y V P A - l a c t o n e (internal  s t a n d a r d , 17 (ig/mL) a n d the precipitate r e m o v e d by centrifugation at 1 3 , 6 0 0 g for 15 min.  T h e supernatant w a s subjected to L C / M S / M S a n a l y s i s .  63  H P L C m e t h o d B a n d M S / M S M R M of transition m/z 4 4 8 -> 1 6 2 w e r e e m p l o y e d for the s e l e c t i v e detection of 5 - G S - 3 - e n e V P A while H P L C method C a n d M R M of transition m/z 6 2 4 - » 4 4 8 w e r e u s e d for 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I.  T h e dwell  times w e r e set at 2 s e c . S t a n d a r d c u r v e s ( r > 0.99) c o v e r e d r a n g e s of 1.0 to 15.0 u. 2  g / m L a n d 3.1 to 50.0 (ig/mL for the mono-conjugate a n d di-conjugate, respectively.  2.5.6.  The ^-glucuronidase catalyzed hydrolysis of the GSH-glucuronides of (E)-  2,4-diene VPA Bile (50 JIL) collected from rats treated with (E)-2,4-diene V P A w a s i n c u b a t e d with p - g l u c u r o n i d a s e (400 units) in p h o s p h a t e buffer (500 u L , p H 5.7) at 3 7 ° C for 15 h. Incubations containing no p-glucuronidase s e r v e d a s control.  T h e reaction w a s  q u e n c h e d by a d d i n g 1 0 % a q u e o u s T F A (50 \xL) a n d the precipitate r e m o v e d by centrifugation at 13,600a; for 15 min. A n aliquot of the supernatant (2 u.L) w a s injected onto the Hewlett P a c k a r d Hypersil O D S c o l u m n a n d eluted u s i n g H P L C m e t h o d C . T h e p r o g r e s s of the hydrolysis w a s determined by following the d i s a p p e a r a n c e of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I by M S / M S in M R M m o d e a s d e s c r i b e d in the p r e v i o u s s e c t i o n (Section 2.5.5)  2.5.7.  Detection and quantitation of the biliary GSH-related conjugates of 4 , 5 -  epoxy VPA and (E)-2,4-diene VPA in 4-ene VPA treated rats Rat bile (100 uL) w a s mixed with a n equivalent v o l u m e of a q u e o u s T F A (0.05%) c o n t a i n i n g 5 - G S - 2 - f l u o r o - 4 - h y d r o x y V P A lactone (internal s t a n d a r d , 17 fig/mL) a n d the  64  precipitate r e m o v e d v i a centrifugation at 1 3 , 6 0 0 g for 15 min.  R a t urine (500 u.L) w a s  a p p l i e d to a C-ig extraction cartridge w h i c h w a s p r e - w a s h e d with m e t h a n o l a n d water. T h e c o l u m n w a s c o n s e c u t i v e l y w a s h e d with water a n d m e t h a n o l . T h e methanol eluate was evaporated  in vacuo  to d r y n e s s a n d the residue re-constituted in a q u e o u s T F A  (0.05%, 100 u,L). A n aliquot of the bile or urine s a m p l e s (2 - 2 0 u l ) w a s injected onto the Hewlett P a c k a r d Hypersil O D S c o l u m n a n d subjected to L C / M S / M S a n a l y s i s . H P L C method A w a s u s e d for chromatographic s e p a r a t i o n of the G S H a n d c y s t e i n y l g l y c i n e conjugates while H P L C method B w a s u s e d for s e p a r a t i o n of the c y s t e i n e a n d N A C conjugates.  M S / M S in M R M m o d e w a s e m p l o y e d for s e l e c t i v e  detection of the following metabolites: 5 - G S - 2 - e n e V P A , transition m/z 4 4 8 - » 123; 5G S - 3 - e n e V P A , transition m/z 4 4 8 -> 162; 5 - G S - 4 - h y d r o x y V P A lactone, transition  m/z  4 4 8 -> 2 8 4 ; 5-cysgly-4-hydroxy V P A lactone, transition m/z 3 1 9 -> 2 8 4 ; 5 - c y s g l y - 3 - e n e V P A , transition m/z 3 1 9 - » 162; 5 - N A C - 4 - h y d r o x y V P A lactone, transition m/z 3 0 4 - » 216;  5 - N A C - 3 - e n e V P A , transition m/z  3 0 4 ->  123; 5 - c y s - 4 - h y d r o x y V P A lactone,  transition m/z 2 6 2 -> 2 4 5 a n d 5 - c y s - 3 - e n e V P A , transition m/z 2 6 2 - » 1 4 1 . T h e dwell t i m e s w e r e set at 2 s e c . S t a n d a r d c u r v e s for quantitation of the biliary metabolites w e r e c o n s t r u c t e d o v e r r a n g e s of 0.5 to 5 0 . 0 u,g/mL with r  2  > 0.99.  T o record full daughter ion s p e c t r a of the metabolites, M S / M S dwell times w e r e adjusted to provide a s c a n rate of ~ 1 s e c / 1 0 0 a m u .  65  2.5.8. Detection and quantitation of the biliary GSH-glucuronide di-conjugate of (E)-2,4-diene VPA in 4-ene VPA treated rats A n aliquot of the bile or urine w a s treated the s a m e a s d e s c r i b e d a b o v e (Section 2.5.7) a n d s u b j e c t e d to L C / M S / M S a n a l y s i s . HPLC  method B a n d M S / M S  in M R M m o d e w e r e e m p l o y e d for  selective  detection of the G S H - g l u c u r o n i d e a n d N A C - g l u c u r o n i d e di-conjugates of ( E ) - 2 , 4 - d i e n e V P A . In combination with the retention times, three transitions w e r e u s e d a s criteria for identification of the metabolites: m/z 6 2 4 -> 4 4 8 , 6 2 4 -> 3 1 9 a n d 6 2 4 -> 162 for 5 - G S 3 - e n e V P A - g l u c u r o n i d e ; m/z 631 - » 4 5 5 , 631 - » 3 2 6 a n d 631 -> 162 for 5 - G S - [ H ] - 3 2  7  e n e V P A - g l u c u r o n i d e ; m/z 4 8 0 -> 3 0 4 , 4 8 0 -> 2 8 6 a n d 4 8 0 -> 123 for 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e a n d m/z 4 8 7 -> 3 1 1 , 4 8 7 ^  2 9 3 a n d 4 8 7 -> 130 for 5 - N A C - [ H ] - 3 -  e n e V P A - g l u c u r o n i d e . T h e dwell times were set at 2 s e c .  2  7  A s t a n d a r d c u r v e for the  quantitation of biliary 5 - G S - 3 - e n e V P A - g l u c u r o n i d e w a s c o n s t r u c t e d o v e r the r a n g e of 3.1 to 5 0 . 0 j i g / m L with r  2  > 0.99.  2.5.9. Alkylation of reduced oxytocin by 2,4-diene VPA-NACA T o oxytocin (1 m g , 1 pmol) in p h o s p h a t e buffer (0.05 M , p H 7.7, 1.0 mL) w a s a d d e d D T T (10 m g , 6 5 fxmol). T h e mixture w a s stirred at ambient temperature for 2.5 h a n d w a s h e d with ethyl acetate.  T o the resultant a q u e o u s solution c o n t a i n i n g the  r e d u c e d oxytocin w a s a d d e d 2 , 4 - d i e n e - V P A N A C A in methanol (15 m g / m L , 120 p L ) , a n d the mixture w a s adjusted to p H 7.7 a n d stirred at ambient temperature for 2 4 h. After acidifying to p H 3.0, the reaction mixture w a s a p p l i e d to a C-|s extraction cartridge  66  w h i c h w a s p r e - w a s h e d with methanol a n d water. w a s h e d with water a n d m e t h a n o l .  The column was consecutively  T h e methanol eluate w a s e v a p o r a t e d  in vacuo  to  d r y n e s s a n d the residue re-constituted in a q u e o u s methanol (1/1, v/v, 0 . 0 5 % T F A , 100 u,L). A n aliquot of the s a m p l e (5 p±) w a s injected onto a P h e n o m e n e x Ultracarb O D S 2 0 guard column  (30 x  1.0  m m , 3 |im)  a n d eluted  into the  LC/MS/MS  using  the  methanol/water ( 0 . 0 5 % T F A ) mobile p h a s e : 7 5 % water for 1 min, a gradient d e c r e a s e to 1 5 % water at 4 min a n d a hold at 1 5 % water for 7 min. T o record a full daughter ion s p e c t r u m of the r e d u c e d o x y t o c i n - 2 , 4 - d i e n e V P A N A C A adduct, the collision g a s w a s at a p r e s s u r e of 6.6 x 1 0 ~ mbar, m a s s resolutions 4  w e r e set at 8.0 for M S 2 a n d the M S / M S dwell time w a s adjusted to provide a s c a n rate of ~ 1 s e c / 1 7 0 a m u .  2.5.10. Alkylation of reduced oxytocin by 2,4-diene VPA-glucuronide T o oxytocin (500 | i g , 0.5 |imol) in p h o s p h a t e buffer (0.05 M , 0.5 m L , p H 7.7) w a s a d d e d tris-(2-carboxyethyl)phosphine (4 m M , 5 0 0 \iL) (Erve et a l . , 1995). T h e mixture w a s stirred at ambient temperature for 15 min. T o the a q u e o u s solution c o n t a i n i n g the r e d u c e d oxytocin w a s a d d e d 2,4-diene V P A - g l u c u r o n i d e in p h o s p h a t e buffer (12.5 |i m o l / m L , 0.5 m L , p H 7.7), a n d the mixture w a s stirred at ambient temperature for 2 4 h. After acidifying to p H 3.0, the reaction mixture w a s a p p l i e d to a C-JS extraction cartridge w h i c h w a s p r e - w a s h e d with methanol a n d water. w a s h e d with water a n d m e t h a n o l .  The column was consecutively  T h e methanol eluate w a s e v a p o r a t e d  in vacuo  to  d r y n e s s a n d the residue re-constituted in a q u e o u s methanol (1/1, v/v, 0 . 1 % formic a c i d ,  67  100 uL).  A n aliquot of the s a m p l e (8 u l ) w a s introduced by direct infusion into the ion  s o u r c e of the L C / M S / M S u s i n g a q u e o u s methanol (1/1, v/v, 0 . 1 % formic acid) a s the mobile p h a s e at a flow rate of 5 0 | i L / m i n . T o detect the alkylated oxytocin, the M S w a s o p e r a t e d at a s c a n rate of ~ 1 s e c / 1 5 5 a m u a n d the multipliers w e r e set at 7 0 0 volts.  2.6. Conjugation of GSH with (E)-2,4-Diene VPA Catalyzed by Rat GST Enzymes  2.6.1. Animals M a l e S p r a g u e - D a w l e y rats ( V a n c o u v e r , B . C . ) w e i g h i n g  1 8 0 to 2 2 0 g w e r e  a l l o w e d free a c c e s s to food ( P u r i n a Laboratory C h o w , P M I Inc., St. L o u i s , M O ) a n d water.  T h e y w e r e h o u s e d in regular c a g e s a n d e x p o s e d to a controlled 12 h c y c l e of  light a n d d a r k n e s s . T w o g r o u p s of rats (6/group) w e r e e a c h administered P B at 7 5 m g / k g p e r d a y for 5 d a y s by i.p. injection.  T h e a n i m a l s w e r e u s e d 2 4 h after the treatment.  Untreated  rats w e r e e m p l o y e d a s controls.  2.6.2. Preparations of rat hepatic mitoplast and cytosolic fractions Rat hepatic mitochondrial ( S h a y i q et a l . , 1991) a n d cytosolic fractions (Siekevitz, 1962) w e r e p r e p a r e d by differential centrifugation. M i t o c h o n d r i a w e r e i s o l a t e d from the p o s t n u c l e a r fraction  a s detailed in the following  s e c t i o n (Section 2.8.4).  Crude  mitochondria w e r e then w a s h e d three times by repeatedly s u s p e n d i n g the pellets in the  68  isolation  buffer  mitochondrial  and  fraction  re-centrifuging  at  w a s estimated  d e h y d r o g e n a s e (LDH) activity  11,000a;. by  C y t o s o l i c contamination  m e a s u r i n g the  (Kline et a l . , 1986).  cytosolic  Digitonin  treatment of the mitochondria (40-50 m g protein/mL) afforded  of  marker  (75 | i g / m g  mitoplasts  the  lactate protein)  which were  further w a s h e d three times with the isolation buffer a n d s u s p e n d e d in s u c r o s e (0.25 M) ( S h a y i q et a l . , 1991).  T h e integrity of resulting mitoplasts w a s e v a l u a t e d by the  liberation of citrate s y n t h a s e , a mitochondrial matrix marker ( S h e p h e r d a n d G a r l a n d , 1969).  M i c r o s o m a l contamination in the mitochondrial a n d mitoplast p r e p a r a t i o n s w a s  determined using potassium cyanide-insensitive N A D P H - c y t o c h r o m e c reductase as the m i c r o s o m a l marker ( L a k e , 1987). T h e mitoplast s u s p e n s i o n w a s centrifuged, 13,600a at 4 ° C for 2 0 min to obtain -  mitoplast supernatant,  or s o n i c a t e d at i c e - c o l d temperature followed by centrifugation,  13,600a; at 4 ° C for 2 0 min to obtain  sonicated mitoplast supernatant.  After s e p a r a t i o n of the mitochondrial pellet, the supernatant w a s centrifuged at 105,000a; for 7 0 min a n d the resulting supernatant c o l l e c t e d a s the A l i q u o t s of the s o n i c a t e d mitoplast  supernatant  i m m e r s e d in boiling water for 4 min to afford  or c y t o s o l i c fraction  boiled subcellular fractions  e m p l o y e d a s controls in the evaluation of G S T e n z y m e activities.  69  cytosolic fraction. were which were  2.6.3. Isolation of the products from the reaction of GSH with 2,4-diene VPANACA in the presence of cytosolic fraction T o a n a q u e o u s mixture (80 m L , 5 % of ethanol, v/v) of 2 , 4 - d i e n e V P A - N A C A (0.41 m m o l , 100 mg) a n d G S H (0.16 m m o l , 5 0 mg) at p H 6.5 w a s a d d e d the cytosolic fraction (0.6 mL).  T h e reaction w a s a l l o w e d to p r o c e e d at 2 5 ° C for 120 min a n d  q u e n c h e d by a d d i n g 1 0 % a q u e o u s T F A (v/v, 5 0 uL).  T h e mixture w a s w a s h e d with  ethyl a c e t a t e , the v o l u m e of the a q u e o u s p h a s e r e d u c e d  in vacuo  precipitate r e m o v e d by centrifugation at 13,600a; for 15 min.  to 2 0 0 u L a n d the  T w o products w e r e  s e p a r a t e d by H P L C : Hewlett P a c k a r d S p h e r i s o r b O D S 2 c o l u m n ;  acetonitrile/water,  1 9 / 8 1 ; flow rate, 1.0 m L / m i n . T h e first fraction containing 5 - G S - ( E ) - 2 - e n e V P A - N A C A eluted at 9.0 min a n d the s e c o n d fraction 5 - G S - 3 - e n e V P A - N A C A at 10.1 min (see S e c t i o n 2.3.12 a n d 2.3.17 for L C / M S / M S a n d N M R data).  2.6.4. GST activity assay The  G S T activities in the s o n i c a t e d mitoplast supernatant a n d the c y t o s o l i c  fraction w e r e m e a s u r e d u s i n g C D N B a s a substrate following the p r o c e d u r e of H a b i g et al. ( H a b i g et a l . , 1974). T o a n a q u e o u s . m i x t u r e of C D N B (1.0 m M ) , G S H (2.5 m M ) , a n d p h o s p h a t e buffer (100 m M , p H 6.7) in a cuvette at 2 5 ° C w a s a d d e d either  the  s o n i c a t e d mitoplast supernatant (10 |iL) or cytosolic fraction (5 p L ) to initiate the reaction.  T h e final v o l u m e w a s 1 m L containing 1% ethanol (v/v).  T h e G S T activity  w a s r e c o r d e d spectrophotometrically by following the i n c r e a s e in a b s o r b a n c e at 3 4 0 nm.  70  2.6.5. GST catalyzed conjugation of GSH with 2,4-diene VPA-NACA The  reaction rate for the addition of G S H to 2 , 4 - d i e n e V P A - N A C A  in the  p r e s e n c e or a b s e n c e of G S T s w a s determined spectrophotometrically. T o a n a q u e o u s mixture of 2,4-diene V P A - N A C A (0.125 m M ) , G S H (2.5 m M ) , a n d p h o s p h a t e buffer (100 m M , p H 6.7) in a cuvette at 2 5 ° C w a s a d d e d o n e of the following: c y t o s o l i c fraction (10 pL), partially purified G S T (100 uL, 2 3 . 7 p g protein) to initiate the reaction. s p o n t a n e o u s reaction involved the boiled cytosolic fraction (TO uL).  The  T h e final v o l u m e  w a s 1 m L c o n t a i n i n g 1% ethanol (v/v). T h e reaction rate w a s r e c o r d e d by monitoring the d e c r e a s e in a b s o r b a n c e at 2 8 4 nm d u e to the d i s a p p e a r a n c e of 2 , 4 - d i e n e V P A NACA.  T h e molar absorption coefficient Ae ( B e r h a n e et a l . , 1994) w a s e s t i m a t e d by  the difference in a b s o r b a n c e v a l u e s between a mixture of 2 , 4 - d i e n e V P A - N A C A / G S H a n d the G S H conjugate products. T h e v a l u e w a s d e t e r m i n e d to be 11.0 m M " c m " . 1  1  T h e catalytic effects of rat liver s u b c e l l u l a r fractions o n the conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A were a l s o m e a s u r e d by c o m p a r i n g the amount of product f o r m e d in e a c h fraction to that of a s p o n t a n e o u s reaction.  T o incubations containing  p h o s p h a t e buffer (100 m M , p H 6.7), G S H (2.5 mM) a n d 2 , 4 - d i e n e V P A - N A C A (0.625 m M ) at 2 5 ° C w a s a d d e d o n e of the following: s o n i c a t e d mitoplast supernatant (300 uL), c y t o s o l i c fraction (300 uL), or partially purified G S T (100 p L , 2 3 . 7 pig protein) to start the reaction.  T h e final v o l u m e w a s 1 m L containing 5 % e t h a n o l (v/v).  reactions involved the c o r r e s p o n d i n g boiled s u b c e l l u l a r fractions  Spontaneous  (300 p L ) .  The  reaction w a s q u e n c h e d at 15 or 3 0 min by a d d i n g 1 0 % a q u e o u s T F A (v/v, 5 0 p L ) a n d  71  precipitates w e r e r e m o v e d v i a centrifugation at 13,600gf for 15 min. A n aliquot of the resultant supernatant (2 (iL) w a s injected onto the P h e n o m e n e x Ultracarb O D S 2 0 c o l u m n a n d eluted u s i n g H P L C method D. M S / M S in M R M m o d e (transitions m/z  549  -> 4 7 4 a n d 5 4 9 -> 4 2 0 for 5 - G S - 3 - e n e V P A - N A C A a n d transitions m/z 5 4 9 -> 2 2 6 a n d 549  123 for 5 - G S - 2 - e n e V P A - N A C A ) w a s u s e d for detection of the p r o d u c t s with the  dwell times b e i n g set at 2 s e c . T h e s t a n d a r d c u r v e s for quantitation w e r e c o n s t r u c t e d o v e r the concentration range of 0.3 to 4 4 . 0 u.g/mL with r The  potential  reaction  of  GSH  with  2  > 0.99.  (E)-2,4-diene  VPA  was  examined  spectrophotometrically at 2 7 2 n m . T h e mixture of (E)-2,4-diene V P A (0.125 m M ) a n d G S H (2.5 m M ) w a s i n c u b a t e d with either rat liver cytosol (10 or 100 u,L) or partially purified G S T e n z y m e s (10 u L , 2 3 . 7 or 2 3 7 u,g protein) in p h o s p h a t e buffer (100 m M , p H 6.7) at 2 5 ° C . T h e final v o l u m e w a s 1 m L containing 1% ethanol (v/v). R e c o v e r y of (E)-2,4-diene V P A from the incubation w a s d e t e r m i n e d by a G C / M S a s s a y ( S e c t i o n 2.2.3).  T h e reaction solution w a s mixed with 0 . 0 5 % a q u e o u s o c t a n o i c  a c i d (w/v) in 1.0 M HCI (300 (iL, internal standard) a n d extracted with ethyl a c e t a t e (5 m L ) . A n aliquot of the ethyl acetate extracts (500 u,L) w a s dried o v e r a n h y d r o u s s o d i u m sulfate, c o n c e n t r a t e d under nitrogen to 100 u l a n d reacted with M T B S T F A (50 |iL) at 6 0 ° C for 1 h to p r e p a r e the t B D M S derivative of (E)-2,4-diene V P A . T h e G C / M S a s s a y w a s c a r r i e d out following a previously reported method ( Y u et a l . , 1995).  72  2.6.6.  In  vitro  formation of the GSH-glucuronide di-conjugates of (E)-2,4-diene  VPA C y t o s o l i c fraction isolated from naive rats w a s u s e d a s the s o u r c e of G S T e n z y m e s (Section 2.6.2). T o incubations containing p h o s p h a t e buffer (0.1 M , p H 6.5) a n d G S H (2.5 mM) in the p r e s e n c e or a b s e n c e of rat liver cytosol (200 u,L) at 2 5 ° C w a s a d d e d 2 , 4 - d i e n e V P A - g l u c u r o n i d e in methanol (16 m M , 2 0 ul.) to initiate the reaction. T h e final v o l u m e w a s 5 0 0 ul_. Incubations that c o n t a i n e d no G S H or cytosol w e r e e m p l o y e d a s controls. The  reaction w a s q u e n c h e d at 3 0 min by a d d i n g 1 0 % a q u e o u s T F A (50 ul.) a n d  precipitates w e r e r e m o v e d v i a centrifugation at 1 3 , 6 0 0 g for 15 m i n . A n aliquot of the resulting supernatant (2 u l ) w a s injected onto the Hewlett P a c k a r d H y p e r s i l O D S c o l u m n a n d eluted using H P L C method A . MS/MS  detection  of  the  G S H - g l u c u r o n i d e di-conjugates  formed  in  the  i n c u b a t i o n s w a s a c h i e v e d v i a M R M of two transitions: m/z 6 2 4 -> 4 4 8 a n d 6 2 4 -> 3 1 9 . T h e dwell times w e r e set at 2 s e c .  2.7. Preliminary Metabolic Studies of a-Fluoro VPA and oc-Fluoro-4-ene VPA in Rats  2.7.1. Animal Experiments S p r a g u e D a w l e y rats ( V a n c o u v e r , B C ) w e i g h i n g 2 4 0 - 2 6 0 g w e r e held in metabolic c a g e s a n d h a d free a c c e s s to food ( P u r i n a Laboratory C h o w , P M I Inc., St.  73  L o u i s , M O ) a n d water.  A q u e o u s solutions of V P A , 4 - e n e V P A , a n d their  fluorinated  a n a l o g u e s (pH 7.0) w e r e administered by a s i n g l e i.p. injection at 1.41 mmol/kg per day for 3 d a y s . U r i n e w a s c o l l e c t e d o v e r a 2 4 h period on the s e c o n d day. B l o o d s a m p l e s w e r e c o l l e c t e d following decapitation, 2 h after the last d o s e , a n d s e r u m s a m p l e s w e r e prepared.  2.7.2. Sample preparations for analysis by high resolution MS and GS/MSD a - F l u o r o V P A a n d a-fluoro-4-ene V P A (50 (ig) w e r e mixed with [ H - | 8 ] B S T F A 2  (50 u.L), respectively.  T h e mixture w a s h e a t e d at 6 0 ° C for 6 0 min to obtain  c o r r e s p o n d i n g [ H - | 8 ] T M S derivatives a n d s u b s e q u e n t l y a n a l y z e d by high 2  the  resolution  M S ( S e c t i o n 2.2.2). Urine a n d s e r u m s a m p l e s (200 p±) were h y d r o l y z e d at p H 12.5, 6 0 ° C for 1 h a n d then acidified to p H 2, or directly acidified to p H 2, a n d extracted with ethyl acetate twice (2 m L e a c h ) . T h e c o m b i n e d ethyl acetate extracts w e r e dried o v e r a n h y d r o u s s o d i u m sulfate, c o n c e n t r a t e d under nitrogen to a final v o l u m e of 5 0 u.L a n d r e a c t e d with either M S T F A or M T B S T F A at 6 0 ° C for 1 h to prepare the T M S a n d t B D M S derivatives, respectively. T h e s a m p l e s w e r e a n a l y z e d using G C / M S D (Section 2.2.3).  74  2.8. Comparative Toxicological Studies of 4-Ene VPA and a-Fluoro-4-ene VPA in Rats  2.8.1.  Animals M a l e S p r a g u e - D a w l e y rats ( V a n c o u v e r , B . C . ) w e i g h i n g 130 to 150 g ( K e s t e r s o n  et a l . , 1984) w e r e randomly a s s i g n e d into g r o u p s of 3-5 a n d a l l o w e d free a c c e s s to f o o d ( P u r i n a Laboratory C h o w , PMI Inc. St. L o u i s , M O ) a n d water.  Rats were housed  in regular c a g e s during the first 4 d a y s then in metabolic c a g e s during the 5th d a y in a controlled 12 h c y c l e of light a n d d a r k n e s s .  2.8.2.  Administration of drugs D o s e s u s e d in the chronic study w e r e s e l e c t e d from p u b l i s h e d d a t a in w h i c h 100  mg/kg of either 4 - P A or 4 - e n e V P A , w h e n d o s e d to y o u n g rats, w a s o b s e r v e d to c a u s e s e v e r 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 ( K e s t e r s o n et a l . , 1984). T h u s , 4 - e n e V P A (0.7 mmol/kg or 100 mg/kg per day) or equivalent oc-fluoro-4-ene V P A (0.7 m m o l / k g or 113 mg/kg per day), 4 - P A (1.0 mmol/kg or 100 mg/kg per day) or equivalent F 2 - 4 - P A (1.0 m m o l / k g or 136 mg/kg per day) w e r e administered in s i n g l e daily d o s e s to  rats  (4/group) for five c o n s e c u t i v e d a y s . A q u e o u s solutions of the s o d i u m s a l t s of the drugs (pH 7.2) w e r e u s e d for i.p. injection. R a t s w e r e fasted 2 4 h prior to their last d o s e a n d urine w a s c o l l e c t e d during this p e r i o d . T h e a n i m a l s w e r e s a c r i f i c e d 1 h after their 6th dose and serum samples were prepared. b e i n g fixed in  Livers w e r e rapidly r e m o v e d with a portion  1% p a r a f o r m a l d e h y d e a n d 2 . 5 % g l u t a r a l d e h y d e  75  in 0.1  M  sodium  c a c o d y l a t e buffer (pH 7.2) for 2 4 h.  T h e remainder of the liver w a s u s e d for the  quantitation of total hepatic a n d mitochondrial G S H . In the acute study, 4 - e n e V P A (1.4 mmol/kg or 2 0 0 mg/kg) a n d equivalent a fluoro-4-ene V P A (1.4 mmol/kg or 2 2 5 mg/kg) w e r e a d m i n i s t e r e d i.p. to rats (9/group), respectively. T h e rats w e r e then sacrificed at 0.75, 2, a n d 4 h p o s t d o s e (3 rats per time point). L i v e r s w e r e quickly r e m o v e d a n d kept ice-cold for the isolation of m i t o c h o n d r i a . C o n t r o l rats in both the acute a n d chronic studies w e r e injected i.p. with normal saline.  2.8.3. Histopathological studies: Rat livers from the chronic treatment groups w e r e subjected to h i s t o p a t h o l o g i c a l examination.  For  light  microscopic  paraformaldehyde/glutaraldehyde  observations  a  section  fixative w a s either s t a i n e d with  of  the  liver  in  hematoxylin-eosin  ( H & E ) , or f r o z e n a n d s u b s e q u e n t l y s t a i n e d with O i l - R e d - O . T h e following criteria w e r e u s e d for s c o r i n g : m i c r o v e s i c u l a r s t e a t o s i s of 0 to 1 0 % of h e p a t o c y t e s , 1+; 10 to 2 5 % of h e p a t o c y t e s , 2+; 2 5 to 5 0 % of h e p a t o c y t e s , 3+; a n d more than 5 0 % of h e p a t o c y t e s , 4+ ( K e s t e r s o n et a l . , 1984). S c o r i n g w a s d o n e blindly. L i v e r s l i c e s from 4 - e n e V P A , a-fluoro-4-ene examined  for  ultrastructural  changes.  Liver  V P A a n d control g r o u p s tissue,  which  was  fixed  were with  p a r a f o r m a l d e h y d e / g l u t a r a l d e h y d e , w a s m i n c e d to 1 m m , fixed in 1% o s m i u m tetroxide 3  in 0.1 M c a c o d y l a t e buffer (pH 7.4) for 1 h a n d in 2 % uranyl acetate solution for 3 0 min, then  dehydrated  with e t h a n o l .  T h e resultant  76  s a m p l e s w e r e e m b e d d e d in  either  e p o n / a r a l d i t e or spurrs. Electron m i c r o s c o p i c e x a m i n a t i o n s w e r e c o n d u c t e d o n ultrathin s e c t i o n s s t a i n e d with lead citrate.  2.8.4. Isolation of mitochondria T h e liver mitochondrial fraction w a s isolated by differential centrifugation at 4 ° C ( P e d e r s e n et a l . , 1978).  Briefly, the rat liver w a s w a s h e d in a Tris-buffer (10 m M  T r i z m a , 5 0 m M s o d i u m chloride) a n d h o m o g e n i z e d in a n isolation buffer (2 m M H E P E S , 7 0 m M s u c r o s e , 2 2 0 m M mannitol, 2 m M E D T A ) .  T h e liver h o m o g e n a t e w a s  centrifuged twice at 1,000p; for 10 min to remove undisrupted t i s s u e , c e l l s , a n d n u c l e i . T h e resultant supernatant w a s centrifuged at 11,000a; for 15 min to pellet mitochondria. C r u d e mitochondria w e r e then w a s h e d three times by repeatedly s u s p e n d i n g the pellets in the isolation buffer a n d re-centrifuging at 1 1 , 0 0 0 g f o r 15 m i n . T h e resultant mitochondrial temperature.  pellet  w a s r e s u s p e n d e d in s u c r o s e m e d i u m  T h e cytosolic contamination  a n d kept  in the mitochondrial  at i c e - c o l d  preparation w a s  e s t i m a t e d from m e a s u r e m e n t of the activity of a cytosolic m a r k e r e n z y m e L D H (Kline et al., 1986) a n d e x p r e s s e d a s the ratio of the total activity in the m i t o c h o n d r i a to that in the h o m o g e n a t e .  T h e respiratory control index a n d A D P / O ratio of the m i t o c h o n d r i a  were  a s a p o o l e d s a m p l e of e a c h treatment group.  determined  Polarographic  m e a s u r e m e n t s w e r e m a d e with a C l a r k - t y p e o x y g e n e l e c t r o d e u s i n g glutamate, in the p r e s e n c e of malate, a s the substrate ( P e d e r s e n et a l . , 1978).  77  2.8.5. Biochemical assays Total hepatic a n d mitochondrial glutathione c o n c e n t r a t i o n s (a s u m m a t i o n of G S H and  G S S G , a n d e x p r e s s e d in G S H thereafter for simplicity) w e r e d e t e r m i n e d by a n  e n z y m a t i c recycling a s s a y immediately after the preparation of the h o m o g e n a t e a n d mitochondria  ( A n d e r s o n , 1985).  T o liver h o m o g e n a t e (500 u.L) or  mitochondrial  s u s p e n s i o n (200 |iL) w a s mixed a n e q u a l v o l u m e of 1 0 % 5-sulfosalicylic a c i d solution. The  mixture w a s vortexed a n d centrifuged in a microcentrifuge (13,600a", 15 min) at  4°C.  A portion of the resultant supernatant w a s p l a c e d in a cuvette with N A D P H a n d  D T N B in p h o s p h a t e / E D T A buffer (pH 7.5). T h e reaction w a s initiated by a d d i n g G R (45 units/mL, 10  T h e amount of G S H w a s determined from the rate of c h a n g e in  a b s o r b a n c e at 4 1 2 n m . T h i s a s s a y w a s found reproducible u s i n g 5-sulfosalicylic a c i d treated s u p e r n a t a n t s w h i c h h a d b e e n stored at - 7 8 ° C for up to 5 d a y s . M i t o c h o n d r i a l G R activity w a s determined by monitoring the c o n s u m p t i o n of NADPH  during  the  reduction  of  GSSG  (Carlberg and  Mannervik,  1985).  The  mitochondrial s u s p e n s i o n w a s s o n i c a t e d o n ice a n d centrifuged in a microcentrifuge ( 1 3 , 6 0 0 g , 15 min) at 4 ° C .  S u p e r n a t a n t (50 \LL) w a s p l a c e d in a cuvette c o n t a i n i n g  water, E D T A , N A D P H a n d p h o s p h a t e buffer (pH 7.0).  T h e reaction w a s initiated v i a  a d d i n g G S S G (20 m M , 100 |iL) a n d the rate of c h a n g e of N A D P H c o n c e n t r a t i o n w a s r e c o r d e d by following the d e c r e a s e in a b s o r b a n c e at 3 4 0 n m . A n o n - s p e c i f i c c h a n g e of NADPH  concentration w a s o b s e r v e d before  adding  GSSG.  T h e r e f o r e , the  GR  activities w e r e reported a s the difference between the two rates a n d e x p r e s s e d a s |i m o l / m i n / m g protein.  78  2.8.6. GC/MS assay of (E)-2,4-diene VPA and the NAC conjugate of (E)-2,4-diene VPA in rats treated with 4-ene VPA or oc-fluoro-4-ene VPA T h e p r e s e n c e of (E)-2,4-diene V P A a n d the c o r r e s p o n d i n g N A C conjugate in the urine a n d s e r u m of rats d o s e d with 4 - e n e V P A or a-fluoro-4-ene V P A w a s e x a m i n e d by the G C / M S D or G S / M S instruments (Section 2.2.3). Urine a n d s e r u m s a m p l e s ( 1 0 - 5 0 u L ) , to w h i c h o c t a n o i c a c i d (10 (ig/mL, 100 (xL) w a s a d d e d a s internal s t a n d a r d , w e r e h y d r o l y z e d at p H 12.5, 6 5 ° C for 6 0 min, then acidified to p H 2 , or directly acidified a n d extracted with ethyl acetate (2 m L ) .  T h e ethyl acetate extracts w e r e dried o v e r  a n h y d r o u s s o d i u m sulfate, c o n c e n t r a t e d under nitrogen to 1 0 0 | i L a n d r e a c t e d with M T B S T F A (50 uL) at 6 0 ° C for 1 h to obtain t B D M S derivatives. F o r qualitative  a n a l y s i s of the N A C conjugate  of ( E ) - 2 , 4 - d i e n e , three ion  fragments, m/z 4 7 4 , m/z 4 1 5 , a n d m/z 2 7 6 , in EI/SIM m o d e w e r e c h o s e n a s criteria for s e l e c t i v e detection of the t B D M S derivative of the N A C conjugate in rat urine.  2.9. Comparative Pharmacokinetic and Metabolic Studies of 4-Ene VPA and a Fluoro-4-ene VPA in Rats  2.9.1. Animals M a l e S p r a g u e - D a w l e y rats ( V a n c o u v e r , B C ) w e i g h i n g 2 4 0 to 2 9 0 g w e r e a l l o w e d free a c c e s s to food ( P u r i n a Laboratory C h o w , P M I Inc., S t . L o u i s , M O ) a n d water.  They  w e r e h o u s e d in regular c a g e s a n d e x p o s e d to a controlled 12 h c y c l e of light a n d d a r k n e s s before the metabolic study.  79  2.9.2.  In  vivo  pharmacokinetic and metabolic study  A group of four rats w e r e administered either a q u e o u s 4 - e n e V P A or oc-fluoro-4e n e V P A (pH 7.0) at 1.41 mmol/kg by i.p. injection. B l o o d (~ 2 0 0 u l ) c o l l e c t e d from the tail v e i n at - 1 5 min a n d at v a r i o u s time points following the d o s e w a s centrifuged at 13,600a; for 2 0 min at 4 ° C to prepare s e r u m s a m p l e s . Urine w a s c o l l e c t e d for 2 4 h. A s e c o n d group of four rats w e r e similarly d o s e d with either of the d r u g s a n d s a c r i f i c e d 1 h post d o s e . T h e liver w a s isolated a n d h o m o g e n i z e d in a T r i s buffer (10 m M T r i z m a , 0 . 9 % s o d i u m chloride, p H 7.4) to give a final v o l u m e of 41 m L . A third group of three rats w e r e a n e s t h e t i z e d with urethane (1 g/kg) a n d their bile ducts c a n n u l a t e d with P E - 1 0 tubing.  Control bile w a s c o l l e c t e d for 15 min.  An  a q u e o u s solution (pH 7.0) of a-fluoro-4-ene V P A w a s then a d m i n i s t e r e d at 0.7 mmol/kg by i.p. injection a n d bile c o l l e c t e d for a n additional 6 h.  2.9.3.  In  vitro  protein binding measurements  4 - E n e V P A a n d oc-fluoro-4-ene  V P A were d i s s o l v e d in water c o n t a i n i n g  an  equivalent of s o d i u m hydroxide, a n d the resultant neutral solutions w e r e e v a p o r a t e d to dryness  in vacuo to  give the s o d i u m salts of the c o m p o u n d s .  T h e s o d i u m salts of 4 - e n e V P A a n d a-fluoro-4-ene V P A w e r e d i s s o l v e d in p o o l e d s e r u m from eight naive rats, respectively, to give c o n c e n t r a t i o n s ranging from 3 5 to 4 0 0 |xg/mL, a n d the s a m p l e s were incubated at 3 7 ° C for 6 0 min. P r o t e i n binding w a s a s s e s s e d v i a ultrafiltration of 4 5 0 u L of e a c h s a m p l e with a n A m i c o n micropartition s y s t e m . E a c h A m i c o n d e v i c e w a s centrifuged at 1 4 0 0 g f o r 15 min at room temperature.  80  Total a n d free s e r u m drug levels w e r e determined by a G C / M S a s s a y d e s c r i b e d in the following s e c t i o n (Section 2.9.4).  2.9.4.  GC/MS detection and quantitation of parent drugs and associated  metabolites (E)-2,4-diene VPA in 4-ene VPA or cc-fluoro-4-ene VPA treated rats Liver h o m o g e n a t e , s e r u m or urine (10 - 3 0 |iL) w a s m i x e d with 100 \iL of o c t a n o i c a c i d ( 1 - 1 0 | i g / m L , internal standard) a n d 100 u l of hydrochloric a c i d (2 M), a n d extracted with 2 m L of ethyl acetate. A s e c o n d set of urine s a m p l e s w e r e either h y d r o l y z e d at p H 12.5, 6 0 ° C for 1 h or incubated with p - g l u c u r o n i d a s e (400 units per 5 0 0 | i L of urine in 0.5 M p h o s p h a t e buffer) at 3 7 ° C , p H 5.7 for 18 h followed by the acidification a n d extraction.  T h e ethyl acetate extracts w e r e dried o v e r a n h y d r o u s  s o d i u m sulfate, c o n c e n t r a t e d to a v o l u m e of 100 u L under nitrogen, a n d h e a t e d with 50 | i L of M T B S T F A at 6 0 ° C for 1 h to p r e p a r e t B D M S derivatives. Quantitative a n a l y s i s of 4 - e n e V P A , a-fluoro-4-ene V P A a n d ( E ) - 2 , 4 - d i e n e V P A w a s c a r r i e d out on the G C / M S D instrument (Section 2.2.3).  T h e a s s a y w a s b a s e d on  E I / S I M of [M-57]+ ions: m/z 199 for 4 - e n e V P A a n d m/z 197 for ( E ) - 2 , 4 - d i e n e V P A , a n d [F-Si(CH3)2]  +  ion: m/z 77 for a-fluoro-4-ene V P A . S t a n d a r d c u r v e s w e r e c o n s t r u c t e d  o v e r c o n c e n t r a t i o n r a n g e s of 1.0 - 6 4 . 0 u.g/mL for 4 - e n e V P A a n d oc-fluoro-4-ene V P A a n d 0.2 - 2.4 p g / m L for (E)-2,4-diene V P A with r  2  > 0.99.  Urinary g l u c u r o n i d e s of the  c o m p o u n d s of interest w e r e estimated from the differences in concentration of parent drug b e t w e e n the h y d r o l y z e d (alkaline or enzymatic) a n d u n h y d r o l y z e d s a m p l e s .  81  2.9.5. LC/MS/MS detection and quantitation of the L-glutamine conjugate in the bile and urine of a-fluoro-4-ene VPA treated rats F o r detection a n d quantitation of the L-glutamine conjugate, rat bile or urine (50 \iL) w a s mixed with a n equivalent v o l u m e of a q u e o u s T F A (0.05%) a n d the precipitate r e m o v e d v i a centrifugation at 13,600a; for 15 min. A n aliquot ( 2 - 5 u,L) of the s a m p l e s w a s injected onto the Hewlett P a c k a r d Hypersil O D S c o l u m n a n d eluted u s i n g H P L C method A . T o record full daughter ion s p e c t r u m of the conjugated metabolite, the M S / M S dwell time w a s adjusted to provide a s c a n rate of ~ 1 s e c / 1 0 0 a m u . Quantitation of a-fluoro-4-ene V P A - G I n w a s c a r r i e d out u s i n g M S / M S in M R M m o d e (transitions m/z 2 8 9 -> 130 a n d 2 8 9 - » 123). T h e ion dwell times w e r e set at 2 s e c . A s t a n d a r d c u r v e , constructed o v e r a range of 4.0 - 6 4 . 0 p:g/mL, h a d r  2  > 0.99.  2.9.6. LC/MS/MS detection and quantitation of the urinary NAC conjugates U r i n e s a m p l e s (2.0 mL) from rats treated with oc-fluoro-4-ene V P A w e r e acidified to p H 2 a n d extracted with ethyl acetate. T h e ethyl acetate extracts w e r e e v a p o r a t e d to d r y n e s s u n d e r nitrogen, the r e s i d u e s d i s s o l v e d in water a n d a p p l i e d to a C - | g extraction cartridge  which  was  p r e - w a s h e d with  methanol  and  water.  The  column  was  c o n s e c u t i v e l y w a s h e d with water a n d methanol. T h e methanol eluate w a s e v a p o r a t e d  in vacuo to  d r y n e s s a n d the residue re-constituted in a q u e o u s T F A ( 0 . 0 5 % , 5 0 u L ) . A n  aliquot (5 u.L) w a s injected onto a P h e n o m e n e x Ultracarb O D S 2 0 g u a r d c o l u m n (30 x 1.0 m m , 3 (im) a n d eluted by a methanol/water mobile p h a s e (1/1, v/v, 0 . 0 5 % T F A ) at a  82  flow rate of 5 0 (iL/min.  T o record full daughter ion s p e c t r u m of 5 - N A C - 2 - f l u o r o - 4 -  hydroxy V P A lactone, M S / M S dwell times w e r e adjusted to provide a s c a n rate of ~ 1 sec/100 amu. T h e urinary N A C conjugates of 4 , 5 - e p o x y V P A a n d (E)-2,4-diene V P A in 4 - e n e V P A treated rats w e r e identified previously (Section 2.5.7). F o r quantitation of the N A C conjugates, rat urine (100 (iL) w a s m i x e d with a n equivalent v o l u m e of a q u e o u s T F A (0.05%) a n d the precipitates w e r e r e m o v e d v i a centrifugation at 13,600gffor 15 min. A n aliquot (2 - 5 u.L) w a s injected onto the Hewlett P a c k a r d H y p e r s i l O D S c o l u m n a n d eluted using H P L C method A .  M S / M S in M R M  m o d e w a s e m p l o y e d for s e l e c t i v e detection of the metabolites: 5 - N A C - 4 - h y d r o x y V P A lactone, transition m/z 3 0 4 -> 2 1 6 ; 5 - N A C - 3 - e n e V P A , transition m/z 3 0 4 N A C - 2 - f l u o r o - 4 - h y d r o x y V P A lactone, transition m/z 3 2 2 -> 2 8 0 .  1 2 3 ; a n d 5-  T h e ion dwell times  w e r e set at 2 s e c . S t a n d a r d c u r v e s w e r e constructed o v e r r a n g e s of 0.5 to 15.0 u.g/mL with r  2  > 0.99.  2.9.7. Pharmacokinetic simulation of the serum data obtained from 4-ene VPA and oc-fluoro-4-ene VPA treated rats D a t a for the s e r u m concentration-time profile of 4 - e n e V P A w e r e fitted using a two compartment m o d e l with a time-lag (Figure 2 . 1 , u p p e r panel) ( S i n g h et a l . , 1988). T h e m a s s b a l a n c e differential e q u a t i o n s for this m o d e l w e r e :  83  dC-|/dt = - ( k  1 0  + k  1 2  )xC-| +k i xA */V-( + k 2  2  x D O S E x e ^ a * / V-|  a  d A / d t = - k i x A * + k-| x C-| x V-) 2  2  2  2  w h e r e C-| is the drug concentration in compartment 1 at time t; A * is the amount of the 2  drug in compartment 2 after time T1 (T1: the time-lag); k-jo is the first-order c o n s t a n t for drug elimination from compartment 1; k-|  rate  a n d k i are the first-order rate  2  2  c o n s t a n t s for the transfer of the drug between the two c o m p a r t m e n t s ; k  a  is the first-  order rate constant for drug absorption into compartment 1; a n d V i is the distribution v o l u m e of c o m p a r t m e n t 1. D a t a for the s e r u m concentration-time profile of a - f l u o r o - 4 - e n e V P A w e r e fitted u s i n g a s i n g l e compartment m o d e l (Figure 2 . 1 , lower p a n e l ) .  The mass balance  differential equation for this m o d e l w a s :  dC/dt = - k  1 0  x C + k  x D O S E x e~ a IV k  a  x  w h e r e C is the drug concentration at time t; k elimination; k  a  is the first-order  is the first-order rate c o n s t a n t for drug  1 0  rate constant for drug a b s o r p t i o n ; a n d V is the  distribution v o l u m e . T h e differential e q u a t i o n s w e r e s o l v e d using the n o n l i n e a r r e g r e s s i o n program M U L T I ( R U N G E ) ( Y a m a o k a a n d N a k a g a w a , 1983).  F o r the s a k e of c o m p a r i s o n , the  s l o p e s of the first linear d e c l i n e s in s e r u m drug concentration-time profiles w e r e defined a s the a p p a r e n t elimination rate c o n s t a n t s (K) a n d the estimation w a s b a s e d o n leasts q u a r e linear r e g r e s s i o n . T h e half-lifes (ty^  84  w  e  r  e  t  h  u  s  c a l c u l a t e d a s 0.693//C. T h e  a p p a r e n t total s e r u m c l e a r a n c e s (CL,p) were determined by the d o s e d i v i d e d by total a r e a u n d e r the s e r u m drug concentration-time c u r v e ( A U C ) .  ki2  Compartment I kv 2  k-io  ka  Compartment I  kio  F i g u r e 2 . 1 . P h a r m a c o k i n e t i c m o d e l s for curve-fitting of the s e r u m concentration-time profiles of 4 - e n e V P A (the u p p e r panel) a n d a - f l u o r o - 4 - e n e V P A (the lower panel), w h e r e k is the first-order rate constant for drug absorption into compartment 1, k-|o is the first-order rate constant for drug elimination from compartment 1, k-| a n d k i a r e the first-order rate c o n s t a n t s for the transfer of the drug b e t w e e n the two c o m p a r t m e n t s a n d T1 is the time-lag. a  2  85  2  2.10. Comparative Pharmacokinetic, Pharmacodynamic and Metabolic Studies of VPA and a-Fluoro VPA In Mice  Animals  2.10.1.  Mature male m i c e ( C D - 1 , C h a r l e s River, P Q ) w e r e h o u s e d in regular c a g e s e x c e p t t h o s e in the metabolic study w h e r e the a n i m a l s w e r e h o u s e d in metabolic c a g e s , a n d a l l o w e d free a c c e s s to food (5015 M o u s e Diet, P M I Inc., St. L o u i s , M O ) a n d water.  Anticonvulsant activity test  2.10.2.  A n t i c o n v u l s a n t activity w a s e v a l u a t e d using the P T Z s e i z u r e t h r e s h o l d  test  ( S w i n y a r d et a l . 1989). T h e s o d i u m salt of V P A or its fluorinated a n a l o g u e in a q u e o u s solution  (pH  7.0)  were  administered  i.p.  to  mice  (8  mice/dose)  at  different  c o n c e n t r a t i o n s . P T Z (85 mg/kg) w a s g i v e n s . c . at different time intervals p o s t d o s e a n d the a n i m a l s w e r e o b s e r v e d for a n additional 3 0 min for s e i z u r e s w h i c h w e r e rated a s : 0, no s e i z u r e ; 0.5, c l o n i c s e i z u r e , animal remained on its feet; 1, tonic-clonic s e i z u r e , a n i m a l lost its b a l a n c e ( L o s c h e r a n d Vetter,  1985).  T h e p e r c e n t a g e of  animals  protected from P T Z i n d u c e d s e i z u r e s w a s plotted against the logarithm of d o s e s to obtain the E D 5 0 of the tested drug. S e i z u r e s o c c u r r e d at a m e a n time of 7.2 ± 4 . 3 min in control m i c e (n = 48) following the administration of P T Z .  86  2.10.3. Preparations of serum, urine and brain samples for analysis of drug concentrations and isolation of brain synaptosomes Mouse  brain s y n a p t o s o m e s w e r e  isolated through  the s e d i m e n t a t i o n  of  a  p o s t n u c l e a r fraction in a d i s c o n t i n u o u s s u c r o s e gradient ( D o d d et a l . , 1981). M i c e w e r e d e c a p i t a t e d into a i c e - c o l d s a l i n e ( 0 . 9 % s o d i u m chloride) at different time intervals after receiving V P A or a-fluoro V P A at 0.83 mmol/kg or 2.08 mmol/kg by i.p. injection. B l o o d w a s c o l l e c t e d a n d the brain r e m o v e d a n d h o m o g e n i z e d in 4 m L of s u c r o s e (0.32 M) c o n t a i n i n g 3 - M P (1 m M , p H 7.0) at 0 ° C .  T h e h o m o g e n a t e w a s m a d e up to a total  v o l u m e of 6.5 m L a n d a 5 0 0 \iL aliquot stored at - 7 8 ° C until a s s a y e d for brain drug concentration.  T h e remainder w a s centrifuged at 1000a; for 10 min, the  resultant  s u p e r n a t a n t a p p l i e d to 9.5 m L of s u c r o s e (1.2 M) a n d centrifuged at 2 2 0 0 0 0 g .  The  fluffy interface w a s c o l l e c t e d , diluted with s u c r o s e (0.32 M) to 10 m L , a p p l i e d to 9.5 m L of s u c r o s e (0.8 M), a n d centrifuged a s before to pellet the s y n a p t o s o m e s . M o u s e urine w a s c o l l e c t e d for 2 4 h following a n i.p. injection of the drugs at 0.83 mmol/kg.  2.10.4. GC/MS quantitation of synaptosomal GABA T h e concentration of m o u s e s y n a p t o s o m a l G A B A w a s d e t e r m i n e d by a G C / M S a s s a y of the c o r r e s p o n d i n g t B D M S derivative (Palaty et a l . , 1994). T h e s y n a p t o s o m a l pellet w a s s u s p e n d e d in 4 . 9 5 m L of a buffer solution (pH 6.4) c o n t a i n i n g p o t a s s i u m c h l o r i d e (0.5 M), s o d i u m p h o s p h a t e (0.4 M), E D T A (10 mM) a n d Triton X - 1 0 0 (0.5%), a n d 2 5 . 9 u.L of [ H e ] G A B A (47.4 nmol in methanol) w a s a d d e d . 2  87  The synaptosomal  s u s p e n s i o n (50 |iL) w a s mixed with 60 fil_ of trichloroacetic a c i d (1.7%) a n d centrifuged at 13600a; for 2 0 min at 4 ° C , a n d 8 0  \iL  portions of the supernatant dried  in vacuo w e r e  h e a t e d with 3 0 0 uL of M T B S T F A ( 1 0 % ) / t B D M S C I (0.1%) in acetonitrile at 6 0 ° C for 1 h to p r e p a r e the t B D M S derivatives of G A B A for G C / M S a n a l y s i s (Section 2.2.3).  The  a s s a y w a s b a s e d on E I / S I M of the [M - 57]+ ions of the t B D M S derivatives, m/z 2 7 4 a n d m/z  2 8 0 for G A B A a n d [ H 6 ] G A B A , respectively. 2  o v e r a range of 0.7 to 2.5 nmol for G A B A with r  2  A standard curve was constructed  > 0.99.  GC/MS detection and quantitation of  2.10.5.  VPA,  a-fluoro  VPA  and the  metabolite (E)-2-ene V P A in mouse brain, serum and urine Brain h o m o g e n a t e (200 u,l_), s e r u m (15 uL) or urine ( 1 5 - 3 0 fiL) w e r e m i x e d with 100 U.L of a q u e o u s o c t a n o i c a c i d ( 1 - 1 0  p g / m L , internal standard) a n d 100 uL of  hydrochloric a c i d (2 M), a n d extracted with 2 m L of ethyl a c e t a t e . A s e c o n d set of urine s a m p l e s w a s h y d r o l y z e d at p H 12.5, 6 0 ° C for 1 h followed extraction.  by acidification  and  T h e ethyl acetate extracts were dried o v e r a n h y d r o u s s o d i u m sulfate,  c o n c e n t r a t e d to a v o l u m e of 100 uL under nitrogen, a n d h e a t e d with 5 0 J I L of M T B S T F A at 6 0 ° C for 1 h to prepare the t B D M S derivatives. T h e G C / M S ( M S D ) a s s a y (Section 2.2.3) w a s b a s e d on E I / S I M of [M - 57]+ ions: m/z 201 for V P A a n d octanoic a c i d ; m/z  199 for ( E ) - 2 - e n e V P A ; [ F - S i ( C H ) ] ion: +  3  2  m/z  77 for a-fluoro V P A . S t a n d a r d c u r v e s w e r e constructed o v e r concentration r a n g e s of 1.0 to 6 4 jj,g/mL for V P A a n d a-fluoro V P A a n d 0.15 to 2.4 p g / m L for ( E ) - 2 - e n e V P A with r  2  > 0.99.  G l u c u r o n i d a t i o n of the drugs in the urine w a s e s t i m a t e d by the  88  differences in concentration of parent drug b e t w e e n the h y d r o l y z e d a n d u n h y d r o l y z e d samples.  2.10.6. LC/MS/MS detection and quantitation of the glutamine conjugate of a fluoro VPA in mouse urine F o r detection a n d quantitation of the L-glutamine conjugate, m o u s e urine (50 u.L) w a s m i x e d with a n equivalent v o l u m e of a q u e o u s T F A (0.05%) a n d the  precipitate  r e m o v e d v i a centrifugation at 13,600a; for 15 min. A n aliquot (2 - 5 uL) of the s a m p l e s w a s injected onto the Hewlett P a c k a r d Hypersil O D S c o l u m n a n d eluted u s i n g H P L C method A . T o record full daughter ion s p e c t r u m of the conjugated metabolite, M S / M S dwell time w a s adjusted to provide a s c a n rate of ~ 1 s e c / 1 0 0 a m u . Quantitation of a-fluoro V P A - G I n w a s carried out u s i n g M S / M S in M R M m o d e (transitions m/z 291 -> 130 a n d 291 -> 162). T h e ion dwell times w e r e set at 2 s e c . A s t a n d a r d c u r v e c o n s t r u c t e d over a range of 2.3 - 7 4 . 0 u,g/mL h a d r > 0.99. 2  2.10.7. Pharmacokinetic simulation of the serum and brain data obtained from a fluoro VPA treated mice T h e p h a r m a c o k i n e t i c parameters for a-fluoro V P A w e r e c a l c u l a t e d u s i n g a two compartment m o d e l (Figure 2.2).  T h e m a s s b a l a n c e differential e q u a t i o n s for this  model were:  89  dC-|/a1 = - ( k  1 0  + k ) x C-i + k i x A / V i + k 1 2  2  2  dA /ot = - k i x A 2  2  2  + ki  a  x D O S E x e'^a* / V i  x Ci x Vi  2  w h e r e C i is the drug concentration in compartment 1 at time t; A  2  is the a m o u n t of the  drug in compartment 2 at time t; k i o is the first-order rate constant for drug elimination from compartment 1; k i  2  a n d k i a r e the first-order rate c o n s t a n t s for the transfer of 2  the drug b e t w e e n the two c o m p a r t m e n t s ; k  a  is the first-order rate constant for drug  a b s o r p t i o n into compartment 1; a n d V i is the distribution v o l u m e of c o m p a r t m e n t 1. T h e e q u a t i o n s w e r e s o l v e d by the M U L T I ( R U N G E ) p r o g r a m ( Y a m a o k a a n d N a k a g a w a , 1983) w h i c h a l l o w e d s i m u l t a n e o u s curve-fitting for two s e t s of e x p e r i m e n t a l d a t a . T h e a p p a r e n t elimination rate c o n s t a n t s of V P A from the m o u s e brain a n d s e r u m w e r e e s t i m a t e d from the s l o p e s of the linear portion of the V P A concentration-time profiles.  ki2 Compartment I  C o m p a r t m e n t II  k2i  (Serum)  J  (Brain)  kio  F i g u r e 2.2. T w o compartment m o d e l for a b s o r p t i o n , distribution a n d elimination of ocfluoro V P A in m o u s e s e r u m a n d brain after a n i.p. d o s e of 0.83 m m o l / k g , w h e r e k i n is the first-order elimination rate constant, k i a n d k i a r e the first-order rate c o n s t a n t s for the transfer of the drug between two c o m p a r t m e n t s , a n d k is the first-order a b s o r p t i o n rate constant. 2  2  a  90  2.11. Protein Concentration Determination  Protein content in s u b c e l l u l a r fractions w a s quantified u s i n g a modified method of Lowry (Markwell et a l . , 1978).  A s t a n d a r d curve of protein c o n c e n t r a t i o n s w a s  g e n e r a t e d u s i n g bovine s e r u m albumin a s a s t a n d a r d .  2.12. Statistics  All quantitative d a t a were p r e s e n t e d a s m e a n ± s t a n d a r d d e v i a t i o n .  A n one-  tailed S t u d e n t ' s n e s t w a s u s e d for statistical c o m p a r i s o n s at a s i g n i f i c a n c e level of p < 0.05.  In c a s e s w h e r e s e v e r a l different treatments w e r e c o m p a r e d with a control,  e s t i m a t e s of the v a r i a n c e w e r e m a d e b a s e d on the the A N O V A (Bolton, 1990).  91  within mean squares  d e r i v e d from  3. Results  3.1. Chemical Synthesis  3.1.1. Synthesis of a-fluorinated analogues T h e F 2 - 4 - P A w a s formed through a R e f o r m a t s k y - C l a i s e n r e a r r a n g e m e n t of allyl chlorodifluoroacetate in the p r e s e n c e of chlorotrimethylsilane. T h e preliminary reaction of allyl chlorodifluoroacetate with chlorotrimethylsilane w a s e s s e n t i a l to e n s u r e the formation of the k e t e n e a c e t a l intermediate, a s p r o p o s e d by G r e u t e r et a l . for the m e c h a n i s m of this reaction (Greuter et a l . , 1988). S i m p l y mixing chlorotrimethylsilane, allyl chlorodifluoroacetate a n d z i n c dust g a v e n o n e of the d e s i r e d product. B a s e d on a report in w h i c h the directed aldol reaction of the lithium enolate of ethyl fluoroacetate w a s d e s c r i b e d ( W e l c h et a l . , 1984), w e attempted to s y n t h e s i z e ocf l u o r o - 4 - e n e V P A a n d 2-fluoro-2-propyl-3-hydroxypentanoic  a c i d (a p r e c u r s o r of oc-  fluoro-3-ene V P A ) through alkylation of methyl 2-fluoropentanoate with allyl bromide and  p r o p i o n a l d e h y d e , respectively ( S c h e m e 3.1).  p r e p a r e d v i a the a c i d c a t a l y z e d methylation  Methyl 2-fluoropentanoate  of 2-fluoropentanoic  was  acid which was  o b t a i n e d from the d e a m i n a t i v e fluorination of norvaline with the hydrofluoride-pyridine reagent ( B a r b e r et a l . , 1982).  Addition of either p r o p i o n a l d e h y d e or allyl b r o m i d e to  methyl oc-fluoropentanoate, however, p r o v e d to be inefficient e v e n in the p r e s e n c e of 1.1  e q u i v a l e n t s of lithium h e x a m e t h y l d i s i l a z i d e , l e a d i n g to r e c o v e r y of the  methyl e s t e r only.  92  starting  NH  (1) H F - P y r i d i n e 2  /NaN0  C H 3 C H 2 C H 2 C H C O O H  2  (2) C H 3 0 H / H+ (Norvaline)  I  CH3CH2CH2CHCOOCH3 (Methyl  2-fluoropentanoate) (1)CH2 = C H C H B r  CH3CH2CHO  2  CH3U / HMDS.  CH3U / H M D S  B  COOH  i (2) H+ / H2O COOH  HO F  (1) CH3SO2CI / DBU (a-Fluoro-4-ene V P A )  (2) H+ / H 2 O COOH  (a-Fluoro-3-ene V P A )  S c h e m e 3 . 1 . S u m m a r y of the attempted s y n t h e s i s of a - f l u o r o - 3 - e n e V P A (pathway A) a n d a - f l u o r o - 4 - e n e V P A (pathway B). H M D S , h e x a m e t h y l d i s i l a z i d e ; D B U , 1,8diazabicyclo[5.4.0]undec-7-ene.  O n the other h a n d , introduction of a fluorine substituent into the a-position of a n aliphatic a c i d c o u l d be a c h i e v e d v i a deaminative fluorination of the c o r r e s p o n d i n g a a m i n o a c i d ( B a r b e r et a l . , 1982).  U s i n g hydrofluoride-pyridine  reagent, d e a m i n a t i v e  fluorination of 2-aminodipropylacetic a c i d , which w a s m a d e by the S t r e c k e r method ( M a r c h , 1985) i n d e e d afforded a-fluoro V P A . Unfortunately, 2 0 % ( b a s e d o n G C / M S a n d H N M R , d a t a not shown) of 2 - e n e V P A (in both E a n d Z forms) w a s o b t a i n e d a s a 1  93  by-product ( S c h e m e 3.2). T h i s by-product probably resulted from the h y d r o - d i a z o n i o elimination of the putative intermediate 2-diazodipropylacetic a c i d , a n d the p r e s e n c e of a n oc-propyl substituent  a p p e a r e d to facilitate  this  elimination  p r o c e s s s i n c e the  d e a m i n a t i v e fluorination of norvaline g a v e only 2-fluoropentanoic a c i d .  CN- / N H  (4-Heptanone)  COOH H+/H 0  3  2  (2-Aminodipropyl acetonitrile)  (2-Aminodipropyl acetic acid) NaNQ  COOH  2  COOH  (2-Ene VPA)  HFPyridine  COOH  (a-Fluoro VPA)  S c h e m e 3.2. Attempted s y n t h e s i s of a-fluoro V P A from 2 - a m i n o d i p r o p y l a c e t i c a c i d which w a s m a d e by the S t r e c k e r method ( M a r c h , 1985).  94  A n alternative synthetic method w a s sought by directly introducing a fluorine into the oc-position of V P A . N F S i w h i c h is a relatively n e w fluorinating reagent reported in the literature (Differding a n d R u e g g , 1991) p r o v e d useful in this reaction.  Following  generation of the lithium enolate of ethyl 2-propylpentanoate u s i n g L D A in the p r e s e n c e of H M P A , the reagent delivers o n e atom of fluorine per m o l e c u l e into the oc-position of the substrate.  T h e p r e s e n c e of H M P A w a s n e c e s s a r y to prevent M i c h a e l addition of  L D A to the vinylic b o n d (Herrmann et a l . , 1973). T h e u s e of H M P A a l s o i n c r e a s e d the solubility of the N F S i reagent at - 7 8 ° C . S u b s e q u e n t l y , a-fluoro-4-ene V P A w a s similarly s y n t h e s i z e d from ethyl 2-propyl4-pentenoate and N F S i . A report of the c o n v e r s i o n of oc,p-unsaturated e s t e r s into the c o r r e s p o n d i n g p,yunsaturated esters ( K e n d e a n d T o d e r , 1982) led us to a p p r o a c h the s y n t h e s i s of ocfluoro-3-ene V P A by u s i n g ethyl 2-propyl-2-pentenoate a s the s u b s t r a t e .  Addition of  N F S i to a T H F solution of the lithium enolate of ethyl 2-propyl-2-pentenoate resulted in ethyl (E)-2-propyl-2-fluoro-3-pentenoate a s the only product w h i c h , u p o n hydrolysis, w a s c o n v e r t e d to the c o r r e s p o n d i n g a c i d .  T h e E form w a s a s s i g n e d d u e to the large  c o u p l i n g constant ( J H H = 17 Hz) o b s e r v e d for the vinylic h y d r o g e n s in the  1  H NMR  s p e c t r u m of the c o m p o u n d . (E)-oc-Fluoro-3-ene V P A w a s found to convert to 2 - p r o p y l - 4 - h y d r o x y - 2 - p e n t e n o i c a c i d lactone s p o n t a n e o u s l y at room temperature.  T h e lactone w a s c h a r a c t e r i z e d from  1H a n d 1 C N M R d a t a a n d from the G C / M S m a s s s p e c t r u m . T h e c o n v e r s i o n is most 3  likely a n intramolecular a c i d c a t a l y z e d p r o c e s s ( S c h e m e 3.3), s i n c e the b a s e hydrolysis  95  of ethyl (E)-2-propyl-2-fluoro-3-pentenoate  at 5 0 - 6 0 ° C for 2 h p r o d u c e d ~ 9 0 % of the  c o r r e s p o n d i n g a c i d w h e r e a s the free a c i d form of (E)-a-fluoro-3-ene V P A c o n v e r t e d to 2-propyl-4-hydroxy-2-pentenoic a c i d lactone within - 1 0 min at 5 0 - 6 0 ° C .  COOH  /  0  "  H  +  F (a-Fluoro-3-ene V P A )  HF  (2-Propyl-4-hydroxy-2pentenoic acid lactone)  S c h e m e 3.3. P r o p o s e d m e c h a n i s m for t h e c o n v e r s i o n of oc-fluoro-3-ene V P A to 4 h y d r o x y - 2 - e n e V P A lactone.  3.1.2. Synthesis of putative GSH conjugated metabolites 3.1.2.1. Synthesis of 5-GS-3-ene VPA ( S c h e m e 3.4).  Esterified 2 , 4 - d i e n e V P A is  k n o w n to b e e s s e n t i a l for the d i e n e to react with G S H ( K a s s a h u n et a l . , 1991), but attempts to obtain the G S H conjugate of the d i e n e f r e e , a c i d by h y d r o l y s i s of t h e ethyl e s t e r h a s led to a facile retro-Michael c l e a v a g e of G S H (Baillie a n d K a s s a h u n , 1994). T h e G S H conjugate of (E)-2,4-diene V P A h a s b e e n p r e p a r e d u s i n g p o r c i n e e s t e r a s e to h y d r o l y z e t h e ester form of t h e G S H conjugated d i e n e ( K a s s a h u n et a l . , 1994). T h e d i s a d v a n t a g e s of this method a r e s e v e r a l c l e a n - u p s t e p s a n d t h e u s e of relatively e x p e n s i v e e s t e r a s e . F o r e x a m p l e , both the G S H conjugate of ( E ) - 2 , 4 - d i e n e V P A a n d the c o r r e s p o n d i n g ethyl ester precursor n e e d e d to b e purified by H P L C ( K a s s a h u n et a l . , 1994). T h u s , a n ideal ester form of (E)-2,4-diene V P A s h o u l d b e s e n s i t i v e towards h y d r o l y s i s at a relatively low p H (at the p H which d o e s not affect t h e stability of G S H  96  peptide bonds) a n d a l s o be a b l e to survive long e n o u g h in a n a q u e o u s solution to c o m p l e t e the reaction with G S H . With this in mind, the trifluoroethyl a n d t B D M S e s t e r s of ( E ) - 2 , 4 - d i e n e V P A w e r e tested a s potential ester c a n d i d a t e s . T h e t B D M S e s t e r w a s s u b s e q u e n t l y found to be labile towards hydrolysis in the a q u e o u s m e d i u m ,  but  trifluoroethyl (E)-2-propyl-2,4-pentadienoate s e r v e d a s a perfect  the  p r e c u r s o r for  conjugation reaction. At p H 10, the conjugation with G S H a n d h y d r o l y s i s to c l e a v e the e s t e r b o n d c a n be s i m u l t a n e o u s l y a c c o m p l i s h e d in o n e run.  T h e relatively high p H  u s e d in this study w a s c o n s i d e r e d to be useful in generating more free thiolate (the p K  a  for G S H <r+ G S " + H+ is 8.66) in the m e d i u m a s well a s facilitating the h y d r o l y s i s of the e s t e r b o n d . T h e tripeptide G S H is stable at this p H for at least o n e w e e k a s d e t e r m i n e d by H P L C (data not s h o w n ) .  GS (Trifluoroethyl 2-propyl -2,4-pentadienoate) S c h e m e 3.4. pentadienoate.  3.1.2.1.  Synthesis  of  (5-GS-3-ene VPA)  5-GS-3-ene  VPA  from  Synthesis of 5-GS-4-hydroxy VPA lactone  trifluoroethyl  ( S c h e m e 3.5).  2-propyl-2,4-  T h e acetone  c a t a l y z e d d e c o m p o s i t i o n of p o t a s s i u m p e r o x o m o n o s u l f a t e p r o d u c e s dimethyldioxirane w h i c h is a very c l e a n a n d efficient reagent for epoxidation (Curci et a l . , 1980).  Upon  mixing with a n a l k e n e , dimethyldioxirane a d d s a n o x y g e n to the d o u b l e b o n d with  97  a c e t o n e b e i n g f o r m e d a s the only by-product. 4,5-epoxypentanoate  In this study, the resultant ethyl 2 - p r o p y l -  c o u l d b e u s e d immediately without further purification.  The  conjugation with G S H a n d hydrolysis of the ester b o n d w e r e a c c o m p l i s h e d in o n e run. S u b s e q u e n t s y n t h e s e s of the c y s t e i n y l g l y c i n e , c y s t e i n e a n d N A C c o n j u g a t e s w e r e c a r r i e d out a c c o r d i n g to similar p r o c e d u r e s .  O II +  H3C-C-CH3  H S O 5  (Peroxomonosulfate)  (Acetone)  O  V /\  COCH2CH3  (Ethyl 2-propyl4-pentenoate)  O O—C COCH2CH3  (Ethyl 2-propyl-4,5epoxypentenoate)  GSH pH 9.5  GS (5-GS-4-hydroxy V P A lactone)  S c h e m e 3.5. S y n t h e s i s of 5 - G S - 4 - h y d r o x y V P A lactone.  3.2. Ionization Constants and the Lipophilicity of VPA, 4-Ene VPA, a-Fluoro VPA and a-Fluoro-4-ene VPA  T h e apparent p K  a  v a l u e s for V P A a n d 4 - e n e V P A w e r e d e t e r m i n e d to b e 4 . 8 0 ±  0.02 a n d 4 . 5 2 + 0.03, respectively, w h i c h are consistent with the literature v a l u e of 4 . 5 6 - 4.8 ( K u p f e r b e r g , 1989).  T h e introduction  98  of a fluorine a t o m , a strong  electron-  withdrawing substituent, into the oc-position of V P A a n d 4 - e n e V P A d e c r e a s e d the p K  a  by m o r e than 1 p H unit, resulting in 3.55 ± 0.03 for a-fluoro V P A a n d 3.24 ± 0.11 for a fluoro-4-ene V P A . U s i n g the H P L C c a p a c i t y factors, the log P v a l u e , a m e a s u r e of the lipophilicity of a m o l e c u l e , w a s found to b e 2.65 for V P A at both p H 4 . 0 a n d p H 2 . 5 , in a g r e e m e n t with the v a l u e of 2.75 obtained by the s h a k e - f l a s k p r o c e d u r e ( K e a n e et a l . , 1983). T h e "log P ' v a l u e for a-fluoro V P A w a s apparently p H - d e p e n d e n t d u e to its relatively lower p K , b e i n g 2 . 0 7 ± 0.04 at p H 4 . 0 a n d 2 . 4 4 ± 0.02 at p H 2 . 5 . S i n c e P is the intrinsic a  partition coefficient of the u n i o n i z e d form of a c o m p o u n d b e t w e e n a n a q u e o u s buffer a n d a n o r g a n i c p h a s e ( J e z e q u e l , S . G . , 1992), the log P v a l u e d e t e r m i n e d at p H 2.5 w o u l d thus b e a better c h o i c e to reflect the lipophilicity of a-fluoro V P A . S u b s e q u e n t l y , 4 - e n e V P A w a s determined to h a v e a log P v a l u e of 2 . 3 5 ± 0 . 0 2 a n d a - f l u o r o - 4 - e n e V P A to h a v e a v a l u e of 2.20 ± 0 . 0 1 .  3.3. Detection and Characterization of Thiol Conjugates in Rats Treated with 4Ene VPA and (E)-2,4-Diene VPA  3.3.1. LC/MS/MS detection of the GSH conjugates of (E)-2,4-diene VPA in the bile of rats treated with (E)-2,4-diene VPA The  detection of the G S H conjugates of (E)-2,4-diene V P A in rat bile w a s  a c h i e v e d u s i n g on-line H P L C s e p a r a t i o n a n d M S / M S monitoring.  Under M R M mode,  two fragments from e a c h c o m p o u n d w e r e e m p l o y e d a s criteria for the identification:  99  transitions m/z 4 4 8 -> 162 a n d 4 4 8 -> 2 5 6 a p p e a r to be d i a g n o s t i c for 5 - G S - 3 - e n e V P A while transitions m/z 4 4 8 - » 123 a n d 4 4 8 -> 2 2 6 are more characteristic for 5 - G S - 2 - e n e V P A (Figure 3 . 1 , A a n d B). T h u s , the two structural i s o m e r s of the G S H c o n j u g a t e s of ( E ) - 2 , 4 - d i e n e V P A w e r e identified in the bile of rats a d m i n i s t e r e d ( E ) - 2 , 4 - d i e n e V P A by c o m p a r i n g the H P L C retention times a n d M S / M S d a u g h t e r ions with the synthetic r e f e r e n c e c o m p o u n d s s p i k e d in control bile (Figure 3 . 1 , C a n d D).  Not o n e of t h e s e  p e a k s w a s d e t e c t e d in blank control bile.  3.3.2. Characterization of GSH-glucuronide di-conjugates in the bile of (E)-2,4diene VPA treated rats As  noted  in the  previous s e c t i o n , L C / M S / M S  s t u d i e s of the  biliary G S H  c o n j u g a t e s of (E)-2,4-diene V P A h a d r e v e a l e d a n u m b e r of unique  fragmentation  patterns a s s o c i a t e d with t h e s e c o m p o u n d s , s u c h a s the d a u g h t e r ion m/z  162 w h i c h is  related to the G S H moiety ( [ H S C H C H C O - g l y ] + ) ( K a s s a h u n et a l . , 1994; S e c t i o n 3.3.1) 2  and  the d a u g h t e r ion m/z  123 which is d e r i v e d from the 2 , 4 - d i e n e V P A portion  ( C H 2 = C H C H = C ( C O ) C H C H C H 3 ) (Section 3.3.1). In a continuing effort to profile the +  2  2  c o n j u g a t e d metabolites of (E)-2,4-diene V P A u s i n g L C / M S / M S , initial Q1 s c a n n i n g for parents of the ions m/z  162 a n d 123 directed our attention to the ions at m/z 6 2 4 a n d  4 8 0 that w e r e present in the bile s a m p l e s . T h e former m a t c h e d the m o l e c u l a r weight of a protonated  G S H - g l u c u r o n i d e di-conjugate of (E)-2,4-diene V P A while the  a p p e a r e d to fit the c o r r e s p o n d i n g N A C - g l u c u r o n i d e di-conjugate.  latter  Subsequent M S / M S  C I D of the parent ions at either m/z 6 2 4 or 4 8 0 p r o d u c e d a neutral l o s s of 176 D a w h i c h  100  w a s indicative of the elimination of the c a r b o h y d r a t e moiety of g l u c u r o n i d e s (Figure 3.2, A a n d 3.3, A ) (Straub et a l . , 1987).  COOH H N^ 2  >  ;OOH  COOH r - ' ^ ^ H - H ,  \XZ  |S  1 +  CONHJ<!  H  >  COOH  256  HN<  C O N H  /N  > d r W  C O O H  H 0,  -d-.  2  162  C C  !  123  2  " •  H  2  ° '  2  2  6  1 \X)NH^COOH N H  MH 10Ch  10Ch  448  162  216 319 256 123  373  u u  ILJLJUJU 100  200  300  400  m/z 500  m/z  i.. .. ,pny, , u^,^ ,, j,. ,, i q^ n  100  tT  T |  200  t  r  (  |  T  300  400  500  F i g u r e 3 . 1 . L C / M S / M S m a s s s p e c t r a of synthetic (A) 5 - G S - 3 - e n e V P A a n d (B) 5 - G S - 2 e n e V P A . T h e configuration of 5 - G S - 3 - e n e V P A r e m a i n s to be clarified.  101  D M R M of 4 Channels E S +  100,  448 > 256  - ^  2 5  2  448 > 256  25.25^  100-s  24.57 0-  r  .  .  r  r  r  y  .  T  V  „  T  r  . j . . ,  ,  T  T  .  r  0  r  25.25^  100l  448 > 162  0  ,,.-,,.p.,. .....|., -,,-,--,,|,, ^^^ r  r r  r  t T  )  T r  l  r  T  25.25^  10Ch  448 > 123  , i  ,  ,  l l  l  ,  ,  25.25^  100-1  , ,  ,  ,  0  ,  448 > 226  •• . , y . , r r  15.00  r  r  r  r ! r  , y,, r  r r T  20.00  ,. ^ . r  r  25.00  r  l  r  f  l  r  r  r  l  i  f  l  24.57  m i ]  r  l  1  448 > 123  1 1 1 1 i i i i 1 | 1 1 ' 1'"i 1 i i T | * r r T T *  24.71  448 > 226  %• 0  f|  r  r  10C  24.7V  448 > 162  •,, .,, . .,„j.,. ii ,,,i,.,...j.... ,. „.|... i.|... .,...,... .,|,i, .,.. „ ,..,|,..|... ... ..  100i  -T T-*i^-T~r r*T* | "t"V'"T"'i' i" r T-T''r'"f"r"T"i "r |" T T" r"r" -  25.25^  100]  30.00  15.00  20.00  25.00  30.00  F i g u r e 3.1 {cont'd). (C) O n - l i n e L C / M S / M S detection of 5 - G S - 2 - e n e V P A (tR = 24.71 min, m/z 4 4 8 -> 123 a n d 4 4 8 - » 226) a n d 5 - G S - 3 - e n e V P A (tR = 2 5 . 2 5 m i n , m/z 4 4 8 - » 2 5 6 a n d 4 4 8 - » 162) in t h e bile of rats treated with (E)-2,4-diene V P A . (D) T h e synthetic s t a n d a r d s s p i k e d in control bile are s h o w n for c o m p a r i s o n .  102  F o l l o w i n g the l o s s of 176 D a , the daughter ion s p e c t r u m of the putative G S H g l u c u r o n i d e di-conjugate g a v e fragments characteristic of G S H c o n j u g a t e s .  A further  l o s s of 7 5 D a (glycine) g a v e m/z 3 7 3 a n d l o s s of 129 D a (pyroglutamate) g a v e m/z 3 1 9 . T h e s e ions plus the daughter ion m/z  162 were e v i d e n c e for the tripeptide g l u - c y s - g l y  b e i n g part of the m o l e c u l e (Figure 3.2, A ) . Additional informative fragments w e r e the ions at m/z 2 1 6 which r e p r e s e n t s R - S - C H - C H = N H 2  al., 1994) a n d m/z  + 2  (R = 3 - e n e V P A ) ( K a s s a h u n et  2 5 6 w h i c h p o s s i b l y results from the c o m b i n e d neutral l o s s of  glutamine, glucuronic a c i d a n d c a r b o n m o n o x i d e (Figure 3.2, A ) . C I D of the parent ion at m/z 4 8 0 p r o d u c e d a prominent fragment ion at m/z  123  w h i c h is likely to a r i s e from the c o m b i n e d neutral l o s s of the N A C moiety, v i a a retroM i c h a e l reaction, plus l o s s of glucuronic a c i d (Figure 3.3, A ) . T h e fragment at m/z 2 9 9 c o u l d result from the l o s s of N A C plus a m o l e c u l e of water while the fragment m/z c o r r e s p o n d s to the l o s s of glucuronic a c i d (Figure 3.3, A ) .  O t h e r d a u g h t e r ions are  p o s s i b l y a s s o c i a t e d with the N A C moiety, including ions at m/z ( [ N A C + H - H 0 ] + ) a n d 130 ( [ N A C + H - H S ] + ) . 2  2  286  164 ( [ N A C + H]+), 146  T h e putative N A C - g l u c u r o n i d e di-  conjugate of (E)-2,4-diene V P A w a s detected in both the bile a n d urine s a m p l e s (Figure 3.4).  103  + 2H, 448/455-  COOH H N<  J ^ > ^ O H  r^^^/Dz  2  CO^H^  CONH^ XOOH  + 2 H , - 176, 3 1 9 / 3 2 6  1001  100]  MH 631  B  455 162 223  100  200 300 400 500 600  m/z  Oi 100  326  *i m / z 200 300 400 500 600  F i g u r e 3 . 2 . M S / M S C I D m a s s s p e c t r a of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I a n d its deuterated a n a l o g u e obtained a s the biliary metabolites in (A) rats treated with (E)-2,4d i e n e V P A a n d (B) a rat d o s e d with [ H 7 ] - 4 - e n e V P A , respectively. T h e fragmentations are d i s c u s s e d in the text. T h e configuration of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I r e m a i n s to b e clarified. 2  104  +  2H, 3 0 4 / 3 1 1 - ,  c-oi  G , 123/130  J  ^  Q  OH  )  [  ^00^  OH  H7/D7  S  CH3CONH COOH MH  10Ch  480  10CH  MH 487 B  304  %-  311 123  4130  )30  462  28(  293|  ^146  )46  p, , ,p.,,,, , qi , T  100  T  i  T  200  \G9  T  r  r  m/z  q. . ^ n  T  300  400  500  0"  \  , „ „ rrp'TT ,.,„p„ . „,,i„ ,, .,, p,, „. „,.| . . ,p„ „ ,,,y. ,.. .,., ,,|,, . ,^ l  100  F  l  t  200  f  T  l  l  T T  t  300  v  >  f  l  f  400  l  f T  T T  m/z  500  F i g u r e 3.3. M S / M S C I D m a s s s p e c t r a of 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e a n d its deuterated a n a l o g u e obtained a s the biliary metabolites in (A) rats treated with (E)-2,4d i e n e V P A a n d (B) a rat d o s e d with [ H y ] - 4 - e n e V P A , respectively. T h e fragmentations are d i s c u s s e d in the text. G represents glucuronic a c i d . T h e configuration of the c o m p o u n d s r e m a i n s to be clarified. 2  105  B MRM  of 3 Channels ES+ 18.02  4 8 0 > 123  17.82  100!  1 ULh  480 > 123  17.72..  4 8 7 > 130  17.72^  487 > 293  % 0  o  TTT'T'TnnnrTTTTTTTTT^r'  10Oi  18.02  i  4 8 0 > 286  " i " 1  1  I mrrT-rmn  17.82  100i  480 > 286  100-|  %-  0  100?  0 yM ,^,. rrr  18.02  i  4 8 0 > 304  rrrr  y. ,, ,, ,|... .. ... ... ,,j rT  ;  r  17.92  100  r  |  |  480 > 304  %4  10Ch  ,  T  ,  r  r  y .  y  T  T  r  r  y ,  r  r  17.92..  r  r  p ,  l  r  T  l  r  4 8 7 > 311  %  Q-^,.......,...|,... ..^.. rri  10.00  0  )  20.00  30.00  10.00  tTrr  ,,,..  fl  20.00  30.00  10.00  r l  . .p T  f T  ,,..|Mp,. . q. ,|» ^  20.00  r  r  r  n  ft  30.00  F i g u r e 3.4. O n - l i n e L C / M S / M S detection of 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e in (A) the bile of rats d o s e d with (E)-2,4-diene V P A (ion transitions for M R M w e r e : m/z 4 8 0 -> 123, 4 8 0 -> 2 8 6 a n d 4 8 0 - » 304); (B) t h e urine of rats d o s e d with 2 , 4 - d i e n e V P A ; a n d (C) t h e bile of rats d o s e d with [ H 7 ] - 4 - e n e V P A (ion transitions for M R M w e r e : m/z 4 8 7 - » 130, 4 8 7 -> 2 9 3 a n d 4 8 7 -> 311) using L C method A . 2  106  E v i d e n c e for the identity of the di-conjugates w a s a l s o o b t a i n e d from the rat d o s e d with [ H ] - 4 - e n e V P A which w a s e x p e c t e d to be m e t a b o l i z e d to [ H ] - ( E ) - 2 , 4 2  2  7  7  d i e n e V P A in vivo. A neutral l o s s of 176 D a w a s o b s e r v e d in the d a u g h t e r ion s p e c t r a of the biliary metabolites, w h e r e the protonated m o l e c u l a r a n d fragment ions c o n t a i n i n g the V P A portion w e r e 7 a m u higher than that s e e n in the bile of rats treated with unlabelled drug. unchanged.  D a u g h t e r ions a s s o c i a t e d with G S H a n d N A C moieties r e m a i n e d  F o r e x a m p l e , fragment ions m/z  162 (Figures 3.2, A a n d B) a n d 146  ( F i g u r e s 3 . 3 , A a n d B) w e r e c o m m o n for the two s e t s of parent i o n s , n a m e l y T h e fragment m/z  624/631 a n d 4 8 0 / 4 8 7 , respectively.  m/z  130 w h i c h a p p e a r s in the  d a u g h t e r ion s p e c t r u m of the parent at m/z 4 8 7 (Figure 3.3, B) m a y represent a merger of two fragments, n a m e l y the hepta-deuterated V P A portion a n d the ion d e r i v e d from [ N A C + H - H2S]+. B a s e d on the M S d a t a a n d the similarity of the C I D fragmentation to that for 5 - G S - 3 - e n e V P A (MH+, 448) ( K a s s a h u n et a l . , 1994; S e c t i o n 3.3.1) a n d 5 - N A C 3 - e n e V P A (MH+, 304) (Section 3.3.3, F i g u r e 3.8, B), the di-conjugates w e r e a s s i g n e d as  5-GS-3-ene  VPA-glucuronide  I  and  1-0-(2-propyl-5-(A/-acetylcystein-S-yl)-3-  pentenoyl)-p-D-glucuronide ( 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e ) , respectively. Further scrutiny of the parent ions at m/z 6 2 4 led to the identification of a s e c o n d G S H - g l u c u r o n i d e di-conjugate of (E)-2,4-diene V P A w h i c h h a d a relatively  shorter  H P L C retention time than that of the apparent 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I. T h e C I D m a s s s p e c t r u m of this c o m p o u n d w a s c h a r a c t e r i z e d by a greater n u m b e r of low a b u n d a n c e fragment ions (Figure 3.5, A ) . A potential structure for this c o m p o u n d w a s a n i s o m e r i c form of the di-conjugate in which a n intramolecular migration of the a c y l  107  moiety b e t w e e n adjacent hydroxyl g r o u p s on the glucuronic a c i d ring h a d o c c u r r e d to g i v e 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II.  T h i s structural a s s i g n m e n t w a s b a s e d on the  o b s e r v a t i o n that the neutral l o s s of 176 D a w a s no longer a dominant p r o c e s s while loss of the glycine a n d glutamate moieties, resulting in the fragments at m/z  549 and  4 9 5 , respectively (Figure 3.5, A ) , a p p e a r e d to be more significant than in the c a s e of 5o G S - 3 - e n e V P A - g l u c u r o n i d e I (Figure 3.2, A ) .  T h e retro-Michael reaction resulting in  the l o s s of G S H w a s a n important p r o c e s s w h i c h , in c o m b i n a t i o n with the l o s s of a water m o l e c u l e , p r o d u c e d the prominent fragment ion at m/z 2 9 9 (Figure 3.5, A ) .  The  most a b u n d a n t daughter ion at m/z 123 w a s likely formed through the c o m b i n e d neutral l o s s of G S H a n d glucuronic a c i d (Figure 3.5, A ) . A d d i t i o n a l e v i d e n c e for the structural identities of the G S H - g l u c u r o n i d e s w a s o b t a i n e d from H N M R s p e c t r a of the H P L C purified metabolites (Figure 3.6). 1  F o r the  H P L C fraction containing the putative 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I ( H P L C fraction 2), the N M R s i g n a l a c c o u n t i n g for two protons at downfield 5.9 ppm is d i a g n o s t i c of the d o u b l e b o n d positioned between the beta a n d g a m m a c a r b o n s of the V P A portion (Figure 3.6, A ) . T h e saturated propyl s i d e c h a i n c a n be e a s i l y identified by a triplet at 0.85 p p m ( C H 3 ) , a multiplet between 1.2 - 1.4 ppm ( C H 2 C H 3 ) a n d a twin multiplet a r o u n d 1.5 - 1.8 ppm ( C H C H 2 ) .  E x i s t e n c e of the G S H moiety is m a r k e d by a  characteristic doublet of doublets at 4.5 ppm ( C y s , C H ) , a twin doublet of d o u b l e t s at 2.75 a n d 2.9 ppm ( C y s , C H ) , a singlet at 4.0 ppm (Gly, C H ) a n d two multiplets at 2.2 2  2  a n d 2.5 p p m , respectively ( G l u , 2 x C H ) . Multiplets at 3.2 - 3.3 a n d 3.5 - 3.6 p p m , a 2  108  doublet at 4.1 p p m ( H O O C C H ) a n d a portion of t h e multiplets at 5.5 - 5.7 p p m a r e c o n s i d e r e d to b e proton s i g n a l s for the glucuronic a c i d ring (Figure 3.6, A ) .  + 2H, 4 4 8 - ^ |  H2O,299 H O G , 12 1 2 3 O.  O  ;OOH  COOH  COOH  H2N-  ^  O  O  H  H  r  H2  G , + H, 226 COiNH< r i C d N H '^ C O O H  CONH  CONH^  ^COOH MH 624  1001  100i  448  >23 2 2 6 319 £ 7 3 I  T  n  100  ,  p  ,  r  ^ ^ ^  200  m  300  /  z  100  4 0 0 5 0 0 600  200  300  400  500 600  m/z  F i g u r e 3 . 5 . M S / M S C I D s p e c t r a of the biliary metabolites of (A) 5 - G S - 3 - e n e V P A g l u c u r o n i d e II a n d (B) 5 - G S - 2 - e n e V P A - g l u c u r o n i d e in rats treated with ( E ) - 2 , 4 - d i e n e V P A . T h e fragmentations for t h e s e c o m p o u n d s a r e d i s c u s s e d in t h e text. G r e p r e s e n t s g l u c u r o n i c a c i d . T h e configuration of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II r e m a i n s to b e clarified.  109  Figure 3.6A.  1  H N M R s p e c t r u m ( D 0 ) of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I i s o l a t e d from 2  the bile of rats d o s e d with ( E ) - 2 , 4 - d i e n e V P A . T h e s i g n a l s w e r e a s s i g n e d a s i n d i c a t e d in the figures. G r e p r e s e n t s proton s i g n a l s from the g l u c u r o n i d e moiety.  110  I L  -Gly  Glu Gly  CU.CH2  Cfcb  i ' '.a  1  • ' i ' 6.5  1  •  1  i  1  6.e  1  •  1  i  1  s.5  1  1  • i  1  s.e  '  1  1  i • • ' i i  n.s  1  > • • i • ».B  1  3.5  PPM  1  1  i >  1  1  3.0  • i • •  1  2.s  1  i  1  •  1  • i > > • • i  2.0  1.5  1  > • • i • i.e  .5  F i g u r e 3 . 6 B . H N M R s p e c t r u m ( D 0 ) of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II i s o l a t e d from 1  2  the bile of rats d o s e d with (E)-2,4-diene V P A . T h e s i g n a l s w e r e a s s i g n e d a s indicated in the figures. *  s i g n a l s a s s o c i a t e d with the putative 5 - G S - 2 - e n e V P A - g l u c u r o n i d e .  proton s i g n a l s from the glucuronide moiety.  Ill  G  represents  D e s p i t e the p r e s e n c e of a n impurity, the N M R s p e c t r u m of 5 - G S - 3 - e n e V P A g l u c u r o n i d e II ( H P L C fraction 1) (Figure 3.6, B) is highly similar to that of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I.  A l t h o u g h the s h a p e s of the p e a k s related to the g l u c u r o n i c a c i d  moiety (3.2 - 3.3 ppm) a p p e a r to b e different, from the limited N M R information it is not c l e a r at the present time to which hydroxyl group in the g l u c u r o n i c a c i d ring the x e n o b i o t i c V P A portion is a t t a c h e d . T h e impurity in H P L C fraction 1 is p o s s i b l y a s s o c i a t e d with the i s o m e r i c 1-0-(2propyl-5-(glutathion-S-yl)-2-pentenoyl)-|3-D-glucuronide ( 5 - G S - 2 - e n e V P A - g l u c u r o n i d e ) , w h i c h c o u l d b e identified in the  1  H N M R s p e c t r u m by a characteristic downfield triplet  at 6.95 p p m ( a - C = C H ) , a r e c o g n i z a b l e sextet at 1.4 p p m ( Y - C H 2 ) a n d a triplet at 2.3 p p m ( 7 - C H 2 ) (Figure 3.6, B, p e a k s with a n asterisk).  O t h e r s i g n a l s w e r e apparently  s u p e r i m p o s e d with t h o s e of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II. P e a k a s s i g n m e n t s for this di-conjugate i s o m e r w e r e largely b a s e d on N M R s p e c t r a o b t a i n e d for 5 - G S - 2 - e n e V P A a n d the c o r r e s p o n d i n g /V-acetylcysteamine thioester of 5 - G S - 2 - e n e V P A ( S e c t i o n 3.4.2, Figure 3.19, B). A M S / M S full daughter ion s p e c t r u m w a s a l s o g e n e r a t e d for 5 - G S - 2 - e n e V P A g l u c u r o n i d e following a proper subtraction from the b a c k g r o u n d d e r i v e d from 5 - G S - 3 e n e V P A - g l u c u r o n i d e II (Figure 3.5, B ) . T h e fragmentation pattern of this di-conjugate w a s s h o w n , u p o n l o s s of the c a r b o h y d r a t e moiety (176 D a ) , to c l o s e l y r e s e m b l e that of 5 - G S - 2 - e n e V P A (MH+, 448) (Section 3.3.1). however, was somewhat ambiguous.  Interpretation  of the C I D m e c h a n i s m ,  F o r e x a m p l e , the a b u n d a n t d a u g h t e r ion at  m/z  2 2 6 c o u l d a r i s e from the c o m b i n e d l o s s of g l y c i n e , pyroglutamate a n d g l u c u r o n i c a c i d  112  a n d the ion m/z  123 from the l o s s of G S H a n d glucuronic a c i d .  O n the other h a n d , it  w a s a l s o p o s s i b l e for t h e s e two ions to result from C I D of the primary d a u g h t e r ion  m/z  4 4 8 following identical fragmentation patterns a s in the c a s e of 5 - G S - 2 - e n e V P A .  3.3.3. LC/MS/MS characterization of the mercapturic acid pathway metabolites in the bile of rats treated with 4-ene VPA T h e C I D d a u g h t e r ion s p e c t r a of the G S H conjugates of 4 , 5 - e p o x y V P A a n d (E)2 , 4 - d i e n e V P A a p p e a r e d to be identical to the reported d a t a ( K a s s a h u n et a l . , 1994, S e c t i o n 3.3.1), feature.  in w h i c h the neutral l o s s of glycine or glutamate r e s i d u e s is a c o m m o n  O n l y a few other fragment ions n e e d to be a d d r e s s e d h e r e b e c a u s e of their  involvement  in the quantitation  of the biliary conjugated  metabolites.  T h u s , the  d a u g h t e r ion at m/z 2 8 4 derived from the protonated 5 - G S - 4 - h y d r o x y V P A lactone w a s likely the result of the c o m b i n e d neutral l o s s of glutamine a n d a m o l e c u l e of water. U p o n C I D , 5 - G S - 3 - e n e V P A p r o d u c e d a fragment ion at m/z s p e c i e s of [ H S C H 2 C H C O - g l y ]  +  162 c o r r e s p o n d i n g to the  ( K a s s a h u n et a l . , 1994), while its structural isomer, 5-  G S - 2 - e n e V P A underwent a facile retro-Michael c l e a v a g e to form a prominent d a u g h t e r ion at m/z 123 (Section 3.3.1). In contrast to the G S H conjugates, C I D of the c y s t e i n y l g l y c i n e c o n j u g a t e s p r o d u c e d no neutral l o s s of 7 5 D a w h i c h c o r r e s p o n d s to a g l y c i n e m o l e c u l e (Figure 3.7).  Interestingly, there w a s a l s o no daughter ion (m/z  244) found to a r i s e from the  c o m b i n e d neutral l o s s of glycine a n d glutamate moieties in the G S H conjugates, indicating that elimination of glycine or pyroglutamate w a s no longer a f a v o r e d p r o c e s s  113  following the l o s s of either o n e of t h e s e two s p e c i e s . A m o n g fragments having c o m m o n m/z  ratios a n d s h a r e d by both 5-cysgly-4-hydroxy V P A lactone a n d 5 - c y s g l y - 3 - e n e  V P A , m/z 2 1 6 m a y represent R - S - C H - C H = N H 2 , w h e r e R = V P A moieties, while ion +  2  m/z  141 is likely p r o d u c e d v i a the neutral l o s s of c y s t e i n y l g l y c i n e r e s i d u e in a retro-  Michael fashion.  A l t h o u g h it w a s s u g g e s t e d that 5 - G S - 4 - h y d r o x y V P A lactone w o u l d  not u n d e r g o a retro-Michael p r o c e s s ( K a s s a h u n et a l . , 1994), the d a u g h t e r ion at  m/z  141 w h i c h p o s s i b l y c o r r e s p o n d s to the protonated 2 - p r o p y l - 4 - h y d r o x y - 4 - p e n t e n o i c a c i d lactone (Figure 3.7, A) h a s b e e n o b s e r v e d in all four of this type of thiol conjugates, n a m e l y , the 5 - G S H , 5-cysteinylglycine, 5-cysteine a n d 5 - N A C substituted 4-hydroxy V P A l a c t o n e s . C o m p e l l i n g e v i d e n c e to support this structural a s s i g n m e n t w a s obtained from C I D of the biliary metabolite derived from [ H 7 ] - 4 - e n e V P A d o s e d to rats, where 2  the transition m/z 3 1 9 -> 141 shifted to m/z 3 2 6 -> 148 (data not s h o w n ) , s u g g e s t i n g that the fragment  is a s s o c i a t e d with the V P A b a c k b o n e .  d a u g h t e r ions w e r e m/z  Additional  informative  177 for 5-cysgly-4-hydroxy V P A lactone, resulting from the  neutral l o s s of 4-hydroxy V P A lactone (Figure 3.7, A ) , a n d m/z  162 for 5 - c y s g l y - 3 - e n e  V P A , r e p r e s e n t i n g the s a m e fragment s p e c i e s s e e n in the c a s e of the c o r r e s p o n d i n g G S H conjugate (Figure 3.7, B) ( K a s s a h u n et a l . , 1994). W h i l e the neutral l o s s of ketene from the protonated 5 - N A C - 4 - h y d r o x y V P A lactone p r o d u c e d a prominent fragment at m/z 2 6 2 (Figure 3.8, A ) , the r e t r o - M i c h a e l c l e a v a g e a p p e a r e d to be a dominant p r o c e s s in the C I D of 5 - N A C - 3 - e n e V P A with the c h a r g e b e i n g retained on either the V P A or N A C moiety, resulting in d a u g h t e r ions at m/z  141 (MH+ - N A C ) , 123 (MH+ - N A C - water) a n d 164 ( N A C + H+) (Figure 3.8, B).  114  T h e fragment ion at m/z  130 o b s e r v e d in both the C I D daughter ion s p e c t r a of 5 - N A C -  4-hydroxy V P A lactone a n d 5 - N A C - 3 - e n e V P A (Figure 3.8) m a y a r i s e from either the protonated N A C following the neutral l o s s of h y d r o g e n sulfate ( N A C + H+ - H 2 S ) or direct c l e a v a g e of the thioether bond on the N A C s i d e of the parent m o l e c u l e .  A  s e c o n d c o m m o n daughter ion w a s ion m/z 216, p o s s i b l y resulting from the c o m b i n e d neutral l o s s of ketene, c a r b o n m o n o x i d e a n d a m o l e c u l e of water, apparently represent the s p e c i e s identical to t h o s e m/z 216 fragment ions derived from the c o r r e s p o n d i n g G S H a n d cysteinylglycine c o n j u g a t e s , respectively, (Figure 3.8). U n d e r the current L C / M S / M S conditions u s e d , very little fragmentation o c c u r r e d following C I D of the c y s t e i n e conjugates. from the protonated 5rcys-4-hydroxy  T h e neutral l o s s of a m o l e c u l e of a m m o n i a  V P A lactone g a v e a daughter ion at m/z  245  (Figure 3.9, A ) , while the neutral l o s s of c y s t e i n e from 5-cys-3-ene V P A v i a a retroM i c h a e l reaction p r o d u c e d a n ion at m/z 141 (Figure 3.9, B ) .  115  COOH  141  s  177  HN  H2N -c-  2  / CONH'  CONH^COOH  T)OOH  + H,162  216 —  MH  MhT 1001  4X3  319  100|  4X3  +  319  B  /o-  162  141 177 216  I 100  141  150  I  |  200  121  284 i  250  300  m/z 350  100  u  150  216 256  200  250  300  m/z 350  F i g u r e 3.7. M S / M S m a s s s p e c t r a of the biliary metabolites (A) 5 - c y s g l y - 4 - h y d r o x y V P A lactone a n d (B) 5 - c y s g l y - 3 - e n e V P A . T h e fragmentations for t h e s e c o m p o u n d s are d i s c u s s e d in the text. T h e configuration of the c o m p o u n d s r e m a i n s to be clarified.  116  o  oCH3COHNK  s 1  1  3  MH  "'}  CH3COHN  0  XOOH  4X2  XX  H 2 0 , 123  s  + 2 H , 262  10G1  COOH  130  COOH  MH H £04  +  +  304  1001  4X2  B  95123 %  po 262  130  95 100  216 162  150  200  250  300  m/z 350  0~<  f  100  m/z 350  p, , .|, .,, . p,j. , ,^ , , .|,. y ,|, , , y. . .. . |. „ .|. , „ .j (  f|  |  |  150  r  <  |  W  t  |  200  f  W  r|  1  r  250  !  1  1  t  W  n  300  n  |  |  F i g u r e 3.8. M S / M S m a s s s p e c t r a of the biliary metabolites (A) 5 - N A C - 4 - h y d r o x y V P A lactone a n d (B) 5 - N A C - 3 - e n e V P A . T h e fragmentations for t h e s e c o m p o u n d s are d i s c u s s e d in the text. T h e configuration of the c o m p o u n d s r e m a i n s to b e clarified.  117  o-  ,0  OOH 141  245_S  HN 2  H N ^ 2  COOH MH H262  COOH MH  +  100i  ix2  10Gi  4X2  +  262  B  %-  95  95  141  24fl 120  123  rrrfrryn'p^rry^TrTTprTTr|"n"rq twjwrffwrrj m/z 100 150 200 250 300  0  i"vvy HTjTTrrjTrrrjTTrrpTri^TTn'yrrnTTi ;  100  150  200  250  m/z 300 rj  F i g u r e 3 . 9 . M S / M S m a s s s p e c t r a of the biliary metabolites (A) 5 - c y s - 4 - h y d r o x y V P A lactone a n d (B) 5 - c y s - 3 - e n e V P A . T h e fragmentations for t h e s e c o m p o u n d s a r e d i s c u s s e d in t h e text. T h e configuration of the c o m p o u n d s r e m a i n s to b e clarified.  118  3.3.4. Quantitation of 5-GS-3-ene VPA and 5-GS-3-ene VPA-glucuronide I in the bile of rats treated with (E)-2,4-diene VPA C a l i b r a t i o n c u r v e s for quantitation of 5 - G S - 3 - e n e V P A a n d 5 - G S - 3 - e n e V P A glucuronide  I were  linear o v e r the  respective r a n g e s of m e a s u r e m e n t s with  coefficients of determination greater than 0.99.  the  T h e coefficients of inter-day variation  w e r e 5 . 2 % a n d 1 1 . 4 % for 5 - G S - 3 - e n e V P A at concentrations of 7.5 (ig/mL a n d 1.0 u. g/mL, respectively (n = 3), a n d 4 . 7 % a n d 5 . 2 % for 5 - G S - 3 - e n e V P A - g l u c u r o n i d e at c o n c e n t r a t i o n s of 5 0 u.g/ml_ a n d 3.1 jig/mL, respectively (n = 4).  T h e coefficients of  intra-day variation w e r e 1.8% a n d 4 . 2 % for 5 - G S - 3 - e n e V P A a n d 1.9% a n d 4 . 8 % for 5G S - 3 - e n e V P A - g l u c u r o n i d e , respectively, at their c o r r e s p o n d i n g c o n c e n t r a t i o n s (n = 3). U p o n administration of (E)-2,4-diene V P A by i.p. injection of ~ 178 u.mol, the biliary excretion of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I a n d 5 - G S - 3 - e n e V P A o v e r a 6 h p e r i o d w a s determined to be 9.9 ± 1.0 (imol a n d 1.9 + 0.2 u m o l , respectively. T h e total biliary G S H - g l u c u r o n i d e di-conjugates, however, c o u l d be in e x c e s s of 10 fimol d u e to the p r e s e n c e of isomeric forms of the metabolites.  T h u s , the s u m of 5 - G S - 3 - e n e V P A  a n d 5 - G S - 3 - e n e V P A - g l u c u r o n i d e s excreted in bile a c c o u n t e d approximately for 6 . 6 % of the d o s e .  119  3.3.5. Detection and quantitation of biliary 5-GS-3-ene VPA-glucuronide I and detection of biliary and urinary 5-NAC-3-ene VPA-glucuronide in 4-ene VPA treated rats L C / M S / M S detection of the di-conjugate 5 - G S - 3 - e n e V P A - g l u c u r o n i d e in the bile of rats treated with 4 - e n e V P A or its hepta-deuterated a n a l o g u e w a s c a r r i e d out using M R M of two s e t s of three transitions (Figures 3.10, B a n d C ) : m/z  6 2 4 -> 448/631  ->  4 5 5 c o r r e s p o n d i n g to a neutral l o s s of 176 D a a p p e a r e d to be characteristic for the g l u c u r o n i d e s while m/z  6 2 4 - » 319/631 -> 3 2 6 a n d 6 2 4 - » 162/631 ->  162 w e r e  indicative of the p r e s e n c e of the G S H moiety in the m o l e c u l e s (Section 3.3.2).  These  transitions c o i n c i d e d at 2 0 . 9 7 min a n d 2 0 . 7 7 min on the H P L C retention s c a l e s for the putative  GSH-glucuronide  respectively. comparison  di-conjugate  and  the  hepta-deuterated  analogue,  T h u s , the c o m b i n e d H P L C retention time a n d M S / M S C I D features, in with  the  authentic  reference  compound,  confirmed  the  structural  a s s i g n m e n t of the biliary metabolite (Figure 3.10). S u b s e q u e n t quantitation of 5 - G S - 3 ene  V P A - g l u c u r o n i d e using the a s s a y d e s c r i b e d earlier ( S e c t i o n s 2.5.5 a n d  3.3.4)  indicated that the excretion of this metabolite in the bile o v e r a 6 h period a c c o u n t e d for 0 . 0 3 % of the 4 - e n e V P A d o s e (Table 2).  120  B MRM of 3 Channels ES+ ,  n  20.77..  n  1UU  624 > 162  l  -j  i  n  _  20.97..  624 > 162 I  1UU - i  I uu —  20.66V.  631 > 162  %i 0-  , , T  100.  2  °-  8  7  ^  6  2  4  >  3 1 9  %  100,  2  r T  ,,  °-  r  9 7  T  T  -  - , - .  T  r  r  0  , . i r  6  2  >  4  (  3 1 9  /o  100-i  20 87,.  624 > 448  Ol.  20.77>.  100-  631 > 326  100  21.07..  624 > 448  0  rTTTTTTTTTTTTTTTTTT-)  100~i  20.77^  631 > 455  /o  /o  010.00  1  % ~ f  T T r T T T - r r ^ y r T T T T T ' r T T ^  TTT rj r rrrjT-rTT-prr-rr-j  T r n ' r m rp i TTTTTTTI  0 20.00  30.00  10.00  |iijin;lj!;f,|. y ff^ - J , 20.00 30.00 T  r  0 10.00  20.00  ft 30.00  F i g u r e 3 . 1 0 . O n - l i n e L C / M S / M S detection of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e in (A) control bile s p i k e d with the reference c o m p o u n d (m/z 6 2 4 -> 162, 6 2 4 - » 3 1 9 a n d 6 2 4 -> 4 4 8 ) , (B) the bile of rats treated i.p. with 100 m g / k g of 4 - e n e V P A (m/z 6 2 4 - » 1 6 2 , 6 2 4 -> 3 1 9 a n d 6 2 4 - » 448) a n d (C) the bile of a rat treated with i.p. with 1 0 0 m g / k g of [ H ] - 4 - e n e V P A (m/z 631 -> 162, 631 -> 3 2 6 a n d 6 2 4 - » 4 5 5 ) . 2  7  121  A  C  B  M R M of 3 Channels ES+ 18.02^  \)} i i i i  480 > 123  /o-  480 > 286  j  n  18.02^  10C  n  100i  480 > 304  h  o  o  100~i  480 > 286  17.92.  ,— ~ —„ „|  RRT  10.00  RRR  RRRR  P[  20.00  » .  YI  30.00  487>130  100:  17.72>.  487 > 293  %0 18.02>  480 > 304  %  »>.«•,»• JL«< •  17.72^ ^  o-  o-  %  0-|„  100-i  %-  UL  o-  480 > 123  o-  rj i i ri ji i i i '|  [  18.02v  10Ch  ^ 17.92^ 100i  n  n  100~i  17.92  i  T  - j  T  T  T  r  y  T  r  487 > 311  v  /o  o- TTTTTTTTTT*'^™rrTTTTT  10.00  A  -(TT"|" i \  20.00  rt 30.00 -  J  10.00  20.00  * „4 L  30.00  F i g u r e 3 . 1 1 . O n - l i n e L C / M S / M S detection of 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e in (A) the bile a n d (B) urine of rats treated i.p. with 100 m g / k g of 4 - e n e V P A (m/z 4 8 0 -> 1 2 3 , 4 8 0 -> 2 8 6 a n d 4 8 0 -> 304), a n d (C) the bile of a rat treated i.p. with 100 m g / k g of [ H ] - 4 e n e V P A ( m / z 4 8 7 -> 130, 4 8 7 - » 2 9 3 a n d 4 8 7 - » 311). 2  7  122  In a similar f a s h i o n , three fragments from e a c h of the N A C - g l u c u r o n i d e a n d its h e p t a - d e u t e r a t e d a n a l o g u e w e r e u s e d a s criteria for the identification conjugate: transition m/z  of this di-  4 8 0 -> 3 0 4 / 4 8 7 -> 311 represent the neutral l o s s of the  d e h y d r o - g l u c u r o n i c a c i d moiety; m/z 4 8 0 -> 2 8 6 / 4 8 7 -> 2 9 3 c o r r e s p o n d to the l o s s of g l u c u r o n i c a c i d a n d m/z 4 8 0 -> 1 2 3 / 4 8 7 -> 130 are likely to a r i s e from the c o m b i n e d neutral l o s s of glucuronic a c i d a n d N A C (Section 3.3.2).  M R M of t h e s e transitions  c o i n c i d e n t at 17.72 min, 17.92 min a n d 17.72 min o n the H P L C retention s c a l e s for the putative biliary a n d urinary N A C - g l u c u r o n i d e di-conjugate a n d for the biliary heptadeuterated a n a l o g u e , respectively (Figure 3.11), illustrates the in vivo formation of 5N A C - 3 - e n e V P A - g l u c u r o n i d e in the m e t a b o l i s m of 4 - e n e V P A .  3.3.6. Quantitation of the mercapturic acid pathway mono-conjugates in the bile  of rats treated with 4-ene VPA Quantitation  of  the  conjugated  metabolites  derived  from 4 - e n e  a c h i e v e d u s i n g on-line H P L C c h r o m a t o g r a p h i c s e p a r a t i o n a n d M S / M S ( F i g u r e s 3.12 a n d 3.13).  V P A was monitoring  Linearity of calibration c u r v e s w a s o b s e r v e d for all a n a l y t e s  o v e r the c o n c e n t r a t i o n s r a n g e s t e s t e d ; coefficients of determination w e r e greater than 0.99.  T h e coefficients of inter-day a n d intra-day variations for the a s s a y of biliary  mercapturic a c i d pathway metabolites w e r e l e s s than 1 5 % at higher c o n c e n t r a t i o n s a n d l e s s than 2 0 % at lower concentrations (Table 1).  123  B MRM of 5 Channels ES+  18.62 10Qi %  448 > 123  %  21.86  I8.62I  0  18.62  100j  18.79  100J  448 > 162  i  18.62 / 2 3  10ft  448 > 284  !  1  1  1 ' ' '  1  1  1  448 > 162  3 9  18.79  100s  448 > 284  %  %'-  !  20.15  100,  °/<H  448>123  21.86  100)  319 > 284  !  '  ' ' I '  ' '  I '  '  '  ' I '  ' '  20.15  100l  18.79k  1  '  I ' '  '  1  I  319 > 284  W  • T r r m r r f T T T r  24.41  100  i **** 1 * * 1 1  10.00  20.00  30.00  319 > 162  1  10.00  20.00  30.00  F i g u r e 3.12. O n - l i n e L C / M S / M S detection of 5 - G S - 2 - e n e V P A {m/z 4 4 8 -> 1 2 3 , t R : 2 1 . 8 6 min), 5 - G S - 3 - e n e V P A (m/z 4 4 8 -> 162, t R : 2 3 . 2 2 min), 5 - G S - 4 - h y d r o x y V P A lactone (m/z 4 4 8 -> 2 8 4 , t R : 18.79 min), 5 - c y s g l y - 4 - h y d r o x y V P A lactone (m/z 3 1 9 -> 284, t R : 2 0 . 1 5 min) a n d 5 - c y s g l y - 3 - e n e V P A (m/z 3 1 9 - » 162, tR: 2 4 . 2 4 min) in (A) t h e bile of rats d o s e d i.p. with 100 m g / k g of 4 - e n e V P A a n d (B) control bile s p i k e d with the synthesized standard compounds.  124  B MRM of 4 Channels ES+  17.98  304 > 216  18.11  100i  304 > 216  17.98 304 > 216 18.79  100i  %ITI T |  17.71^  10CH  0- ^  I I I I | I I I I | I I I I |  304 > 123  ». .py.. M ..^.. ... ,,,(, .|. r ,, rr  |  ry  ra)  i  l  l  f  18.93,  1 Q  lfllVl  rr—-r—r—  . , .| 1  l  304 > 123  18.93  %-J  9  M?  18.931  10Ch  304 > 123  /o~ i ,.. , „p.»,,. i| ,i | r  r  li!  f  |  l  lllri  , | | | i ii i ii i:| l  ll  |ll  l  18.66  (  f  f  - - - y^- - -p- - y:. .,,i, .yi^  f  rT  262 > 245  T  T  rn  r  r1  )  r  18.11  10&3  0  r  •i  262 > 245  rrprrrT f"ri»T'r|' 'i"rrir{ ,  ,  18.79  ^20.29  %•  0 20.29.  262 > 141  II  . .. . ^,.. , , ,„p „ „1„ J„ ,„ „ j  T ?  100-  r r  100  r r r7  rT  rlT  18.11  1  lf  rlr  r  10.00  T  „ , . .  r  T  T  .  r  20.00  r  r  .  r  r  r  r  n  rt  262 > 141 100  30.00  10.00  T 20.00  I I J ny., nn ,y, „ i,y |,^ ii „y„ ,| r  y  r  7  r T  M  M  t  r  l  1  %-i r  262 > 245  "'M rt 30.00  20.2SL.  262 > 141  J  " ^ - T T ' T T - p r T ' T T - J ' T - r T T - p r T ' T T ^ ft  10.00  20.00  30.00  F i g u r e 3 . 1 3 . O n - l i n e L C / M S / M S detection of 5 - N A C - 4 - h y d r o x y V P A lactone ( m / z 3 0 4 - » 2 1 6 , t : 1 7 . 9 8 min), 5 - N A C - 3 - e n e V P A (m/z 3 0 4 1 2 3 , t R : 1 8 . 9 3 min), 5 - c y s - 4 hydroxy V P A lactone ( m / z 2 6 2 -> 2 4 5 , t R : 18.66 min) a n d 5 - c y s - 3 - e n e V P A ( m / z 2 6 2 -> 1 4 1 , t R : 2 0 . 2 9 min) in (A) the bile a n d (B) urine of rats d o s e d i.p. with 1 0 0 m g / k g of 4 e n e V P A , a n d (C) control bile s p i k e d with the s y n t h e s i z e d s t a n d a r d c o m p o u n d s . R  U p o n administration of 4 - e n e V P A by i.p. injection at 1 0 0 m g / k g , t h e biliary excretion of G S H related m o n o - c o n j u g a t e s d e r i v e d from 4 , 5 - e p o x y V P A a n d (E)-2,4d i e n e V P A o v e r a 6 h period w a s estimated to b e 6.3 u,mol a n d 1.24 u,mol, respectively ( T a b l e 2). T h u s , the s u m of the mercapturic a c i d pathway metabolites a c c o u n t e d for 4 . 8 % of the d o s e .  125  A m o n g the biliary metabolites, the N A C conjugates of 4 , 5 - e p o x y V P A a n d (E)2 , 4 - d i e n e V P A w e r e detected in the urine a s well (Figure 3 . 1 3 , B ) . N o effort w a s m a d e to c h r o m a t o g r a p h i c a l l y resolve s t e r e o i s o m e r s of the c o n j u g a t e d metabolites.  Table 1. Coefficients of inter-day and intra-day variations for assay of biliary mercapturic acid pathway metabolites (n = 3) Compounds  3  Concentration (M-g/mL)  Inter-day  Intra-day  C.V.  C . V . (%)  D  (%)  5-GS-4-OH V P A lactone  20.6 2.6  6.4 3.3  2.0 3.9  5-GS-3-ene VPA  7.5 1.0  5.2 11.4  1.8 4.2  5-cysgly-4-OH V P A lactone  25.4 3.2  2.8 10.6  3.3 6.7  5-cysgly-3-ene VPA  12.7 1.6  12.7 13.7  2.9 11.1  5-NAC-4-OH V P A lactone  5.5 0.3  6.9 7.3  1.4 7.3  5-NAC-3-ene VPA  5.0 0.3  12.1 10.5  1.5 3.6  5-cys-4-OH V P A lactone  20.0 1.5  7.1 11.5  4.7 1.0  5-cys-3-ene VPA  7.5 0.5  7.0 5.8  3.8 6.3  a MS/MS in MRM mode was used for quantitation: m/z 448 -> 284, 5-GS-4-hydroxy VPA lactone; m/z 448 -> 162, 5-GS-3-ene VPA; m/z 319 -> 284, 5-cysgly-4-hydroxy V P A lactone; m/z 319 -> 162, 5-GS-3-ene VPA; m/z 304 -> 216, 5-NAC-4-hydroxy V P A lactone; m/z 304 -> 123, 5-NAC-3-ene VPA; m/z 262 -»245, 5-cys-4-hydroxy VPA lactone and m/z 262 -> 141, 5-cys-3-ene VPA. b C.V.: coefficient of variation.  126  CD  'c  v>  0  (0  o —. d^ o  ^  1_ « ^  o ©  +l  1  I CO  v  in O  1_  o  SI  o X  <  to  >  "5  CD  c <u w CO  S2 c c (0 x 3  !»  o— d d +1 ^ 4-1  ^ d  O  $2  d  CD  0 0  as CD CD  c  CD •4—•  3  CO CD  o  o  .CO  CD  o  ^  I  CNJ O  d ^ d c£ +i  21  CD  m  oZ £  Is  © re < O a> CD LO § CD o  0-  •a  O  ° o UJ -o £  5 -a £ «  =5 o a> 15  CD CO  '  +1  CO  •*rr  1  w  CO  re c E CO  2 re« C«D /i\  CD -Q CO C TJ CO CD CD C _D k Q. co E 9X X S ? O CD o O CD CD TJJ LO Q- CO  =1 CO  £  1?  co  o c  9o >^ o > c ®  to o  o o -o  o —. d ^ -t-i 1X1  -t-i co  CD  >*  J-  CD  CO  CO  CD J) Q_  LO  2-  ~ CO TJ ^  ~ E  CD O CD  in CD re ^  n S TJ ^ re re -5 CD  CD T-  O < o . o - o_ , ' <• X5 1  CL « < > CD Q_ CD 1  D) 2 C TJ e >, re x :  0  -I  3  o re CD  E  E t o 0  o  D)  >, «  -— — i  1 ^  E 9>  CO  I co  o  E CD §_  T3  a> re  +1 LO  d^ 3  +1 CNJ  So  X  CO  o  < >  CO  o  E  -Q  o  CD  HI •  0-  X  Q.  if)  11 _1  CO  T3 re CD > N =• <  ~ £ >  C/)  o  CO •-  $ CD  CD  c  CD  9< CM I  ^  07  c c re <p | o CO  re X DC ^5 a. CC  127  TJ O ~ £ ^ CD c C 0_ CD o « (D "D T3 O < CO CO ©  > reX O < §^  O X} D_ CD >  re  +-«  o — i  c  re -  I g.|  re  *= ?T  •  CD O  re c  re re 2, co 'to re si k_  re o  E re c o  T3  re o re o en T3 CD CO O T3 CD  o  CD T3  E  -ST  CD  2 re  o  "E* o u  II  1  0 5  o CO  jg  <i ^  E < o co o z:  re *  +1  d  c 'o  re  re l-  5  CD —  o  CM Q)  1  re _  cr <° re c c  ±= CD  CD 4-1  in  El  ^  (D  Q.  CO  < -2  O  ^  CO  CD 3  0) V  CD  o o  o  +-»  < >  c o  D *Cu  = (D CO o © O I— LO re m o  O 0 O)  re +-» c  CD iO CD 0_  3.3.7.  Incubation of the biliary 5-GS-3-ene VPA-glucuronides with (3-  glucuronidase F o l l o w i n g incubation of the rat bile with p - g l u c u r o n i d a s e for 18 h at p H 5 . 7 , the L C / M S / M S M R M signals recognized a s 5 - G S - 3 - e n e VPA-glucuronide I were decreased significantly in c o m p a r i s o n with the control incubation containing n o e n z y m e , while the p e a k s c o r r e s p o n d i n g to 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II r e m a i n e d u n c h a n g e d (Figure 3.14, C ) . T h e r e w a s n o difference between the 0 a n d 18 h i n c u b a t i o n s w h e n pg l u c u r o n i d a s e w a s a b s e n t (Figures 3.14, A a n d B ) .  B MRM of 2 Channels ES+ 29.81 624 > 319 100i  29.54  100-j  624 > 3 1 9  100  27.63.  624 > 319  27.77 .  624 > 448  %4  10Ch  29.81  624 > 448  100n  100  0 V°y^f-T'rT-yV-"R"T ) TT ft 11 11  20.00  04  s  40.00  20.00  40.00  20.00  40.00  F i g u r e 3.14. O n - l i n e L C / M S / M S detection (ion transitions for M R M w e r e : m/z 6 2 4 -> 3 1 9 a n d m/z 6 2 4 -> 4 4 8 ; L C method C ) of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I (tR: 2 9 . 8 min) a n d 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II (tR: 2 7 . 7 min) in (A) after 0 min a n d (B) after 18 h of incubation c o n t a i n i n g the bile of rats treated with (E)-2,4-diene V P A , a n d (C) after 18 h of incubation of the s a m p l e with p - g l u c u r o n i d a s e .  128  3.3.8. Alkylation of reduced oxytocin by 2,4-diene-NACA and 2,4-diene VPAglucuronide B e c a u s e the esterified forms of (E)-2,4-diene V P A (either O- or S - esters) w e r e o b s e r v e d to react with G S H (Section 2.3.17 a n d S e c t i o n 2.6.6), their  reactivities  t o w a r d s biological peptides or proteins w e r e investigated u s i n g r e d u c e d oxytocin a s a m o d e l substrate.  W h e n r e d u c e d oxytocin w a s a l l o w e d to mix with 2 , 4 - d i e n e V P A -  N A C A , L C / M S d a t a clearly indicated the formation of a £>/s-adduct w h i c h g a v e the protonated m o l e c u l a r ion at m/z 1491 (Figure 3.15, A ) . E v i d e n c e w a s a l s o o b t a i n e d to s u g g e s t the p r e s e n c e of a d o u b l y - c h a r g e d alkylated peptide with the ion at m/z  746.  T h e ion at m/z 7 5 7 m a y represent the doubly c h a r g e d m o n o - s o d i u m salt of the adduct (Figure 3 . 1 5 , A ) . S u b s e q u e n t C I D of the protonated b/s-adduct (MH+: m/z  1491) (Figure 3.15, B)  r e v e a l e d a similar fragmentation pattern to that reported for a c a r b a m o y l a t e d m o l e c u l e of oxytocin ( P e a r s o n et a l . , 1991): the product ions a p p e a r to a r i s e from the c l e a v a g e of a m i d e b o n d s in the peptide b a c k b o n e . F o r e x a m p l e , c l e a v a g e b e t w e e n r e s i d u e s C y s - 6 a n d P r o - 7 results in the prominent daughter ion at m/z  1207 w h i c h retains both of the  di-alkylated r e s i d u e s . T h e counterpart ion at m/z 2 8 5 c o m p r i s e s the protonated form of the first three C-terminal a m i n o a c i d r e s i d u e s . A n o t h e r major fragment at m/z  6 2 9 is  most likely p r o d u c e d by the c l e a v a g e b e t w e e n A s n - 5 a n d C y s - 6 with c h a r g e retention o c c u r r i n g o n the mono-alkylated C-terminal portion (Figure 3.15, B ) . T h e s e key ions p r o v i d e d u n a m b i g u o u s e v i d e n c e for the alkylation of C y s - 6 .  T h e s e c o n d site for  alkylation is p o s s i b l y a s s o c i a t e d with the N-terminal C y s - 1 or the T y r - 2 r e s i d u e , b a s e d  129  o n the p r e s e n c e of a daughter ion at m/z  5 0 8 w h i c h is representative of the m o n o -  alkylated peptide with c l e a v a g e occurring b e t w e e n Tyr-2 a n d lle-3.  H o w e v e r , the  M S / M S experiment failed to discriminate to w h i c h residue, C y s - 1 or T y r - 2 , the 2 , 4 - d i e n e V P A - N A C A was bound. Similarly, L C / M S e v i d e n c e indicated the formation of a rj/s-adduct w h e n r e d u c e d oxytocin w a s a l l o w e d to mix with 2,4-diene V P A - g l u c u r o n i d e (Figure 3.16). T h e ions at m/z 1641 a n d 821 c o r r e s p o n d to the s i n g l e c h a r g e d a n d d o u b l e c h a r g e d protonated dialkylated peptide, respectively. Other p e a k s a s s o c i a t e d with the b/s-adduct a r e the ion m/z  832  representing the  double charged mono-sodium  adduct  of the  alkylated  oxytocin a n d the ion at m/z 1465 p o s s i b l y resulting from the neutral l o s s of 176 D a . In addition to the di-alkylation, the M S spectrum r e v e a l e d the p r e s e n c e of a m o n o - a d d u c t , forming a protonated m o l e c u l a r ion at m/z 1325 (Figure 3.16).  130  A  10Os  b/'s-Adduct (M + 2 H )  2 +  746  b/'s-Adduct MH 1491 +  822 i  L  700  800  900  1000  B2 H N - CYS - TYR 2  I  R  B3  1100  B4  ILE -i G L N  Y7"  Y6"  1200  B5 ASN  Y5"  1300  B6 CYS I  1400  B7  1500  B8  P R O i- L E U i-GLY - N H  2  --R Y 4 " " Y3"  MH  B 100  m/z  +  H 1491  <x3  B6  1207 Y4" B3 629 621. ' Y5" B4 Y6" 743 870 Y7" 700 984  200  400  600  800  1000  B8 1 1 8 9  \  1417  1200  1400  tm/z  F i g u r e 3.15. (A) L C / M S m a s s spectrum of a £>/s-adduct formed u p o n reaction of 2,4d i e n e V P A N A C A with r e d u c e d oxytocin. (B) T h e alkylation w a s d e t e r m i n e d to o c c u r at the free c y s t e i n e r e s i d u e s a c c o r d i n g to the daughter ion s p e c t r u m of the £>/'s-adduct. R: A/-acetyl-S-(2-propyl-3-pentenoyl)cysteamine.  131  H N - C Y S - T Y R - ILE - G L N - A S N - C Y S - P R O - L E U - G L Y - N H 2  I  2  I  R  R  i»/s-Adduct (M + 2 H ) 100  2 +  821  % b/s-Adduct 176 mono-Adduct +  b/'s-Adduct 1465  0 800  MH  +  1641 •n-r m/z  1000  1200  1400  1600  F i g u r e 3.16. L C / M S m a s s spectrum of the mixture of products formed u p o n reaction of 2 , 4 - d i e n e V P A - g l u c u r o n i d e with r e d u c e d oxytocin. T h e alkylation w a s p r e s u m e d to o c c u r at the free c y s t e i n e r e s i d u e s . R represents 3-ene V P A - g l u c u r o n i d e .  132  3.4.  Conjugation of GSH with (E)-2,4-Diene VPA Catalyzed by Rat Liver GST  Enzymes  3.4.1. GST activities in rat liver subcellular fractions T h e cytosolic fraction a n d the supernatant of s o n i c disrupted mitoplasts isolated from rat liver w e r e a s s a y e d for G S T activities using C D N B a s a substrate. T h e activity w a s d e t e r m i n e d to be 1533 ± 104 nmol/min/mg protein in the cytosolic fraction, w h i c h is c o n s i s t e n t with the  reported  value  of  1400 nmol/min/mg  protein  (DePierre  and  M o r g e n s t e r n , 1983). F o l l o w i n g P B treatment, the cytosolic G S T activity w a s i n c r e a s e d to 2 0 6 % of control (Table 3). T h i s elevation in G S T activity lies within the 1 . 6 - 3 fold r a n g e of i n c r e a s e s previously s e e n in rats pretreated with P B ( A n i y a et a l . , 1 9 9 3 ; Foliot a n d B e a u n e , 1994). Microsomal  contamination  in  the  crude  mitochondria,  as  measured  by  c y t o c h r o m e c r e d u c t a s e activity, w a s 5 . 0 % a n d d e c r e a s e d to < 1.0% after the digitonin treatment  a n d three  subsequent washes.  The  specific activity  mitochondrial preparations w a s 2 . 1 % of that in the cytosolic fraction.  of  L D H in  the  This cytosolic  contamination in the mitoplast fraction w a s c o n s i d e r e d to be r e d u c e d to ~ 0 . 4 % or l e s s following the digitonin treatment a n d three w a s h e s . T h e mitoplasts, w h i c h w e r e judged to be intact, s h o w e d > 9 8 % of citrate s y n t h a s e activity following s o n i c disruption.  133  Table 3. GST activities in rat liver subcellular fractions Treatment  Subcellular  Total activity  fraction  (nmol/min/ml_)  3 0  c  S p e c i f i c activity (nmol/min/mg of protein)  % increase (specific activity)  Untreated  Cytosol  Untreated  Mitoplast supernatant  2.44 ± 0.36  N.D.  Untreated  Sonicated mitoplast supernatant  12.29 + 0.52  111 ± 5  0  3162 ± 9 2  106  1533 ± 1 0 4  P B treated  Cytosol  P B treated  Mitoplast supernatant  1.40 ± 0.32  N.D.  P B treated  Sonicated mitoplast supernatant  2 3 . 3 0 ± 0.74  161 ± 5  Partially purified GST  0  45  17281 ± 8 9 0  a. Rats were dosed i.p. with an aqueous solution of PB at 75 mg/kg for 5 days. The animals were used 24 h after the final dose of PB. Untreated rats were employed as controls. b. Rat liver subcellular fractions were isolated by differential centrifugation. Partially purified GST enzyme was obtained from Sigma Chemical Co. (St. Louis, MO). c. The GST activities were determined spectrophotometrically at 25°C using CDNB as a substrate. Each incubation (1.0 mL) contained GSH (2.5 mM), CDNB (1.0 mM), rat liver mitochondrial fraction (10 |iL) and phosphate buffer (100 mM, pH 6.7). The values represent averages of 2 - 3 different experiments (mean ± S.D.). d. Each incubation contained GSH (2.5 mM), CDNB (1.0 mM), rat liver subcellular fraction (10 |j.L) and phosphate buffer (100 mM, pH 6.7). The values represent averages of 2 - 3 different experiments (mean ± S.D.). N.D.: not determined.  134  T h e total G S T activity in the s o n i c a t e d mitoplast supernatant w a s e s t i m a t e d to be  5-fold higher than that in t h e mitoplast supernatant (Table 3 ) .  Following P B  treatment, G S T activity in the s o n i c disrupted mitoplasts w a s i n c r e a s e d 8 9 % , no e l e v a t i o n w a s s e e n in t h e e n z y m e activity a s s o c i a t e d with t h e intact (Table 3).  whereas mitoplasts  T a k e n together, t h e s e results s u g g e s t that t h e G S T activity that w a s  d e t e c t e d in t h e s o n i c a t e d mitoplast supernatant h a d b e e n e n c a p s u l a t e d within t h e mitochondrial matrix ( A d d y a et a l . , 1 9 9 4 ) .  3.4.2. Characterization of the products formed by conjugation reactions T h e s p o n t a n e o u s reaction between G S H a n d 2 , 4 - d i e n e V P A - N A C A resulted in o n e product (Figure 3 . 1 7 , A , tR = 2 0 . 2 9 min) w h i c h s h o w e d a s i g n a l in the  1  H NMR  s p e c t r u m at 5 . 5 p p m representing 2 vinyl protons a n d therefore w a s identified a s 5 - G S 3 - e n e V P A - N A C A (Figure 3 . 1 8 , A ) .  A l t h o u g h the d o u b l e b o n d in the c o r r e s p o n d i n g  free a c i d , n a m e l y 5 - G S - 3 - e n e V P A , w a s previously a s s i g n e d the  trans  configuration  ( K a s s a h u n et a l . , 1 9 9 1 ) , it a p p e a r e d that the major 5 - G S - 3 - e n e V P A - N A C A i s o m e r h a d the cis configuration s i n c e the c o u p l i n g constant of the two vinyl protons ( J H H = 7 Hz) w a s d e t e r m i n e d to b e m u c h s m a l l e r than that reported for the G S H c o n j u g a t e of (E)2,4-diene  V P A ( J H = 1 6 Hz). H  A n u m b e r of s m a l l s i g n a l s e m e r g i n g from t h e  b a c k g r o u n d in the a r e a of 5 . 3 5 - 5 . 5 5 p p m c o u l d b e indicative of a trace a m o u n t of the  trans  isomer.  In addition to 5 - G S - 3 - e n e V P A - N A C A , a s e c o n d G S H c o n j u g a t e of 2 , 4 -  d i e n e V P A - N A C A w a s isolated from reactions involving either the rat liver mitoplast o r c y t o s o l i c fractions  (Figure 3 . 1 7 , B a n d C , t p = 1 8 . 7 9 min).  135  This  isomer w a s  c h a r a c t e r i z e d through its  1  H N M R s p e c t r u m by a downfield (6.6 ppm) triplet s i g n a l  c o r r e s p o n d i n g to a s i n g l e vinyl proton c o u p l e d with the adjacent - C H 2 - , the a b s e n c e of a n a-proton s i g n a l (at 3.2 ppm a s in the c a s e of 5 - G S - 3 - e n e V P A - N A C A ) a n d a large downfield shift (~ 1 ppm) of the p" methylene protons to 2.3 ppm (Figure 3.18).  This  G S H addition product w a s a s s i g n e d the structure 5 - G S - 2 - e n e V P A - N A C A . The LC/MS/MS  m a s s s p e c t r a of the two  G S H c o n j u g a t e s a p p e a r e d to  be  dissimilar, but still s h a r e d s e v e r a l c o m m o n fragments w h i c h w e r e featured by ion p e a k s of M H + (m/z  549), M H + - g l y c i n e (m/z  474), M H + - pyroglutamic a c i d (m/z  420), and  M H + - g l y c i n e - pyroglutamic  a c i d - A/-acetylcysteamine (m/z  P r o m i n e n t d a u g h t e r ions at m/z  123 m a y result from a r e t r o - M i c h a e l reaction (loss of  226)  (Figure  3.19).  G S H ) a c c o m p a n i e d by the c l e a v a g e of the thioester b o n d (loss of A/-acetylcysteamine). A c o m b i n e d neutral l o s s of glycine, A/-acetylcysteamine a n d c a r b o n m o n o x i d e w o u l d g i v e the d a u g h t e r ions at m/z 3 2 7 w h i c h , following the further l o s s of pyroglutamic a c i d , convert to the d a u g h t e r ions at m/z  198 (Figure 3.19).  O t h e r informative  fragments  i n c l u d e m/z 2 5 6 resulting from the c o m b i n e d l o s s of glutamine, A/-acetylcysteamine a n d c a r b o n m o n o x i d e in the c a s e of 5 - G S - 3 - e n e V P A - N A C A (Figure 3.19, A ) a n d m/z  355  c o r r e s p o n d i n g to M H + - glycine - A/-acetylcysteamine in the c a s e of 5 - G S - 2 - e n e V P A N A C A (Figure 3 . 1 9 , B ) .  136  B MRM of 4 Channels ES+ 20 29  549.00 > 474.00  100i  10Ch  CM  CM 20.29  100i  0y'"TT~i  549.00 > 420.00  20.29  c4 ;  i i  i  | i i i  i  | 1 1 t 1  1  20.43  10.00  CH T  20.00  549.00 > 226.00  i  ] i i  i  '"I!Ir'T  , ! !  g""l  r~f~1  T i  i |-T"T"i  i I—Ir'"i—|-"i  r  549.00 > 420.00  p  r-rn-T-T-r-T-  20.29  10CH  CH T—r—r-i j " ' " ' r - r " T — r — i  i |  549.00 > 474.00  20.15  10Ch  f~T-T-r-T-T-T-r-T-r-i—p-r—T—T—T—|  100  20.15  549.00 > 123.00  r'T"T""i  20.29  30.00  20.00  549.00 > 226.00  r-r—i  r  1  549.00 > 123.00  30.00  F i g u r e 3 . 1 7 . L C / M S / M S c h r o m a t o g r a m s of (A) 5 - G S - 3 - e n e V P A - N A C A (tR = 2 0 . 2 9 min, m/z 5 4 9 -> 4 7 4 a n d 5 4 9 -> 4 2 0 ) obtained from the s p o n t a n e o u s reaction of G S H with 2 , 4 - d i e n e V P A - N A C A ; (B) 5 - G S - 2 - e n e V P A - N A C A ( t = 18.79 min, m/z 5 4 9 - » 2 2 6 a n d 5 4 9 -> 123) a n d 5 - G S - 3 - e n e V P A - N A C A (tR = 2 0 . 2 9 min) from t h e mitoplast p r o m o t e d reaction. A l l e n z y m e c a t a l y z e d reactions of G S H a n d 2 , 4 - d i e n e V P A - N A C A p r o c e e d e d at p H 6.7 for 3 0 min w h e r e a s the s p o n t a n e o u s reaction w a s c a r r i e d out at p H 6.7 for > 10 h, in a q u e o u s p h o s p h a t e buffer solutions. R  137  D MRM of 4 Channels ES+ 549.00 > 474.00  20.29  lOOi  20.02  1001  18.79  18.66 \  \  20.29  10Ch  549.00 > 420.00  20.15  100  18.79 0-  100n  "r"»""r"T  111  18.79  [  i""T"  ••r"'T r""v ,m  549.00 > 226.00  10Cb  ,20.29  100l  549.00 > 420.00  18.66 \  | !"-r-r 1 | 1 IT ;  -r~r-T—r—f—1—1—r  549.00 > 474.00  r  p r-r—T—T—r-r-r  i'"''r'"'i'""r—)- T T™r"»""'ir"T""i a  m  18.66  r  549.00 > 226.00 .20.15  %i 0-  1  18 93 549.00 > 123.00 .20.43  1003  20.15 18.7SC  549.00 > 123.00  04  10.00  20.00  30.00  F i g u r e 3 . 1 7 (cont'd). L C / M S / M S c h r o m a t o g r a m s of (C) t h e products from t h e c y t o s o l promoted reaction; a n d (D) t h e products from t h e partially purified G S T c a t a l y z e d reaction. A l l e n z y m e c a t a l y z e d reactions of G S H a n d 2 , 4 - d i e n e V P A - N A C A p r o c e e d e d at p H 6.7 for 3 0 m i n .  138  -NACA COSCH2CH2NHCOCH3 CH2CH=CHCHCH Cys  2CH2CH3  !  SCH2CHCONHCH2COOH I HCOCCHCH2CH2CONH 1  NH  :  2  Glu-  -Gly  ; J  NACA COCH  Gly CH  2  Glu CH  CH2Chb SCfcbCH =CHChL NACA CH  CH=CH  NACA CH 2  2  Glu CH  Glu  2  Cys  Jl I 6  1  1  5  '  5-5  0  CH  2  CHCtb  CU2CH3  L  LA.  • I 6  3  1  5.0  4.5  1  1  1  ' 1  3.5  4!o  3.0  PPM  20  2.5  15  10  r-NACA COSCHjO^NHCOCHa CHzCHzCH^CCHjCH^Ha  B  Cys:" £ SCH2CHCONHCH I H O O C C H C H ^ ^ N H I I -Gly NH : Glu-  2COOH  2  J  Gly CH  NACA  2  COCH3  Glu CH  CHJJCHJ  NACA CH 2  =CCH , Glu CH 2  2  NACA CH  SCh CH , Cys CH 1 2  2  CH=C  1  Ctl2CH= Glu CH  Cb2CH  2  1  6.0  5.5  • 1' 5.0  • 1' 4.0  • 1 3.5 PPM  3  A  UlJLJ 6.5  F i g u r e 3.18.  2  Cys CH  1  2  '  1  3.0  2.5  2.0  1.5  I ' 1.0  .5  H N M R s p e c t r a ( D 0 ) of (A) 5 - G S - 3 - e n e V P A - N A C A a n d (B) 5 - G S - 2 2  e n e V P A - N A C A isolated from the rat liver cytosolic fraction promoted conjugation  of  GSH  as  with 2 , 4 - d i e n e V P A - N A C A .  T h e s i g n a l s were a s s i g n e d to the structures  indicated. 139  QOS^NHCOCH  I - W M J  COOH H 2 N -<  3  J - N A C A , 123  ^CdNH^  L  + 2H.420  :  COS^NHCOCHa JZ^  COOH H N -f  J - N A C A , 123  2  VrtN^ i  ^  S  ^CdNH^COOH  C  T^CdNH^COOH i  474—'  N A C A , 226  J  F i g u r e 3.19. M S / M S m a s s s p e c t r a of (A) 5 - G S - 3 - e n e V P A - N A C A a n d (B) 5 - G S - 2 - e n e V P A - N A C A isolated from the rat liver cytosolic fraction promoted conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A . Fragmentation patterns of the two c o m p o u n d s are d i s c u s s e d in the text. N A C A represents A/-acetylcysteamine. T h e configuration of the c o m p o u n d s r e m a i n s to be clarified.  140  L C / M S / M S quantitation of the G S H conjugates formed during the incubation of 2 , 4 - d i e n e V P A - N A C A with G S H in the p r e s e n c e of rat liver s u b c e l l u l a r fractions w a s b a s e d on M R M of transitions of m/z 5 4 9 H> 4 7 4 a n d m/z 5 4 9 - » 123. C a l i b r a t i o n c u r v e s s h o w e d g o o d linearity with the coefficient of determination greater than 0.99.  The  coefficients of inter-day variation were 1.5% a n d 4 . 6 % for 5 - G S - 2 - e n e V P A - N A C A at c o n c e n t r a t i o n s of 4 0 (ig/mL a n d 2.5 | i g / m L , respectively, a n d 3 . 4 % a n d 4 . 8 % for 5 - G S 3 - e n e V P A - N A C A at concentrations of 4 4 u.g/mL a n d 2.8 u.g/mL, respectively (n = 3). T h e coefficients of intra-day variation w e r e 1.9% a n d 3 . 4 % for 5 - G S - 2 - e n e V P A - N A C A a n d 5 . 4 % a n d 5 . 3 % for 5 - G S - 3 - e n e V P A - N A C A , respectively, at their c o r r e s p o n d i n g c o n c e n t r a t i o n s (n = 3). A l t h o u g h both G S H conjugates of 2 , 4 - d i e n e V P A - N A C A w e r e d e t e c t e d in all reactions involving either the rat liver cytosol or mitoplasts, the ratios of the products w e r e distinctly different d e p e n d i n g on the s u b c e l l u l a r fractions. of 5 - G S - 3 - e n e V P A - N A C A  to 5 - G S - 2 - e n e V P A - N A C A ,  F o r e x a m p l e , the ratio  determined  by  quantitative  L C / M S / M S a n a l y s i s of either the 15 or 3 0 min incubation, w a s 1.17 - 1.47 for the reactions involving untreated a n d P B treated c y t o s o l , but w a s 5.8 - 8.7 for the s o n i c a t e d mitoplast supernatant promoted conjugation (Table 4).  Partially purified G S T e n z y m e  g a v e a product ratio of 2.0 w h e n it w a s u s e d for the incubations (Table 4).  141  3.4.3. Catalytic effects of rat liver subcellular fractions on the addition reaction of GSH and 2,4-diene VPA-NACA T h e reaction rate b e t w e e n G S H a n d 2,4-diene V P A - N A C A c o u l d be monitored spectrophotometrically by following the d e c r e a s e of a b s o r b a n c e at 2 8 4 nm d u e to the d i s a p p e a r a n c e of 2 , 4 - d i e n e V P A - N A C A .  In rat liver cytosolic fraction the conjugation  rate i n c r e a s e d 7 8 7 % , a s c o m p a r e d with the incubation containing b o i l e d c y t o s o l .  An  additional 1.5-fold e n h a n c e m e n t w a s s e e n in the cytosolic fraction isolated from P B treated rats (Table 4). S i m i l a r results w e r e obtained w h e n the reaction w a s followed by L C / M S / M S quantitative a n a l y s i s of the products, the s u m of 5 - G S - 2 - e n e  VPA-NACA  a n d 5 - G S - 3 - e n e V P A - N A C A b e i n g 2 3 1 1 % of control with a further 1.7-fold i n c r e a s e after  P B treatment  (Table  4).  It w a s  not  f e a s i b l e to  measure  reaction  spectrophotometrically for incubations involving the s o n i c a t e d mitoplast b e c a u s e of interference from the supernatant. conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A .  rates  supernatant  H o w e v e r , this fraction did promote the T h e L C / M S / M S a s s a y indicated that  the G S H c o n j u g a t e s of 2,4-diene V P A - N A C A w e r e i n c r e a s e d by - 1 0 0 % w h e n the s o n i c a t e d mitoplast supernatant w a s included in the incubations (Table 4).  N o further  elevation of G S T activity towards the reaction of G S H a n d 2 , 4 - d i e n e V P A - N A C A w a s d e t e c t e d in the mitoplasts isolated from P B treated rats (Table 4). Partially purified rat liver G S T e n z y m e w a s o b s e r v e d to c a t a l y z e the conjugation in a similar f a s h i o n to that of isolated cytosolic a n d mitoplast fractions in terms of the a p p a r e n t e n h a n c e d conjugation reaction rates, a n i n c r e a s e d production of the G S H c o n j u g a t e s , a n d the formation of two isomeric products (Figure 3.17 a n d T a b l e 4).  142  g  T3  CD CO CO CD o c .!=  c £ £  c o o  o 3  o  i -  o  CM CM  T -  CO CO  Q  •  CO CO CD CD  c CO  *CO E c  as  CO c  o CL  3 O  CD  3  3  O uCT to CO  u  c CQ 'CD -I—»  o  •g CL  Q.I  o  a  „,  LO  co ^ £ 2 , LO CM CO o d d  +i ,  o E  E  l<  c  „  o  +i  +i  00 CO CO  aj  CM  O  O  £2- £2CO d  CO  +1  +1  lo  CO T-  1  CM  LO  CD CO  d  +i CD  o  o  £2- £2oo d  co d +i +i I - CO 00 00 S  m" i5  CO  °- §-  CD d LO +1 00  co CM  T3 CD *S ~ to CO >0  CD  CD CD O  O  CO CO  ill  E  •4—'  CD d  CO  +1  CM  T CM CO  cd  +1  LO  O +1 00 d  CO i d d +1 +1 O CO  - i -  LO d CM +1  CO CO LO CO  c  o  "•4—•  CO CO  o 1—  o  CD  o  o CD •4—>  rr  Q. E  |  "fr  o  CL CO  2  TJ  CD  •4—*  CO +-» CO  CO  o co o  £ o -i—•  o  E  Q-  T3 T J O <D  O CQ CO O  "O "CD CD +2 CO CO CD CD i= 3  CQ Q_  O co  O co  c  o  % O  o  s,  O  CO C i_D  -+—<  c  3 CO  -*—»  CO _CC CL O  -t—»  0) *  CD  0_  "to to  CL  o  H CO  o CD  I CO  '•c  CO  a.  143  0 E o  '  o CL o o 0 CL co  C  o E 0 i_  0  O  <  1  5  > 0  £V. "° "a uo 0 0  0  CO  -»—• re o o to 0 c o to 0 E  o a c CO .— CD H -a i — )= 0 CO  •Q 3 to  0 O  E  •=  = Mco re co DC 0 . 0 .  co  T3 CO  o S re 'o < TJ re o 0  2  CO 0 > CO  < o <  CL E 02 LO to 0 CM. _3  re > 0  2  0 o -° O O ZZ- c: ^- . .  CO  0 CO  o  +—>  ° x co CQ -O  CD D)  °-'c  3 ^ o CO LU  §  CO 0 o T3 0 o LO CM  to -*—>  d  U .  H — «4—  2  *  E  «  < re 831-« O J0) ^  4—"  0 X2  E  >  I—  c 0 0  T J CO  0 +1 < k_ C CL 0 re 0 >  c 0 CM Q- E 0 "n: 0 <p E CO LO CO CM  0 CM" c ' « CD "O TJ c o CO o X X CL 7= CO 00 C ^  c g  <  0 CO 2 c0 o3 < TJ TJ CL 4 O  L-  o  re  5  •4 0c CM to 0  X CO  E l  3 LO CM TO" J CO CO C  o  0 T3  CO  |  < to CL 0 > CD  C  7j5  S i  < CM  0  re w  3 0 o TJ TJ 0VI/ w 0 c re * ; c: re >- re o C  cr LO 0 CM 0  0  -i  re  s E  O  LO  CM  CD - CL 0  <  ^  o to c 0  i_  E CO CD  to c o  T3 CD  o 0 >  i2 ® <  ro  E c  CO  E  5  o  "CD o _Q 3 C7)  ICM +1 00 CO CO S  CM CM d d +1 +1 CO LO CD  o d +1 LO d  o <  _3  'c—  TJ  c  J5  CD "CO  •4—'  3^  'c  .2 jB 6  c 0  c 'CD  o  •r = o  o  %  0  ?  c  O  CO  00 CM  • CD W  .5  c  © *2 CO o O to ^ CD x : co  2  re  o  CO _ E o re S  P -  <  to  o  o  o  to 0 CD  5 re  > 0 Q  9  ^ 0  O CM 0 E  0 >  re re  CL W JD 0 C  -r  1 o  CO  >  m  2  CL CL  TJ  2  IT  ^ re > 0  9 § «8 LO •<= 0  .. re "5  "  =i-  o O CO  sz  c g  re 3 O c 0  CO  0 3 c "E _c 0 E 0 x••—» : 0 re g TJ _c CO  ^—' 0 o re  i_ 0  re re  N o c h a n g e in a b s o r b a n c e at 2 7 2 nm w a s detected w h e n the reaction of G S H and  (E)-2,4-diene  V P A was  monitored  spectrophotometrically  during  a  30  min  incubation c o n t a i n i n g either the cytosolic fraction or partially purified G S T , a n d did not c h a n g e following a 10-fold i n c r e a s e in the concentration of G S T e n z y m e .  Further  confirmation that no reaction h a d taken p l a c e b e t w e e n G S H a n d the d i e n e free a c i d w a s o b t a i n e d from the G C / M S semi-quantitation of (E)-2,4-diene V P A r e c o v e r e d from the incubation m e d i u m . T h e p e a k a r e a ratio of (E)-2,4-diene V P A to internal s t a n d a r d w a s d e t e r m i n e d to be 2.2 ± 0 . 1 for a 0 min reaction in buffer a n d 2.2 ± 0.1 after 3 0 min of incubation with 2 3 7 u.g of protein of partially purified G S T .  T h e s e d a t a are the  a v e r a g e s of two determinations d o n e in duplicate.  3.4.4. In vitro formation of the GSH-glucuronide di-conjugate of (E)-2,4-diene VPA A p r o c e d u r e d e s c r i b e d for isolation of V P A - g l u c u r o n i d e (Williams et a l . , 1992) w a s a d o p t e d to extract 2,4-diene V P A from the bile of rats treated with (E)-2,4-diene VPA.  T h i s p r o c e d u r e w a s effective in eliminating most of the u n - r e a c t e d parent drug  a n d e n d o g e n o u s c o m p o u n d s excreted in the bile.  Further purification through flash  c h r o m a t o g r a p h y afforded 2,4-diene V P A - g l u c u r o n i d e .  C I D of the c o m p o u n d w a s  d o m i n a t e d by the neutral loss of 176 D a characteristic for g l u c u r o n i d e s .  144  B M R M of 2 Channels ES+ 18.33  100| %* 0-  18.12  624 > 319  0-  -TTj-rr-r-rjT-T-TTjr-rT-rr^ 1 8.43  100a  1  624 > 448  ,, ,,, n ,,|,,,,, . ,. y .. .. ^- - ^- . .|, ,,,) ,1 | , i  f  (  r r  r  rT  T  t  t  rr  rTT r  18.22  100i  %  624 > 319  624 > 448  %, , ,p,, ,, ,j„, r  10.00  20.00  rrr  TT  r  30.00  rrT  rt 30.00  . ,,p ^ t  r  10.00  20.00  D 18.53  100,  624>319  624 > 319  %• _A^.*«-i*^ iA/ ***** >  0  <  . . ..y. . . .^^^  rT p t  18.53  100i  624 > 448  T r v  624 > 448  10Oi %  rii^fT^..".. " vj-h .i , y. , .^ 10.00 T  r  rT  rrr  56  r  20.00  rt 30.00  , . r  T m |  -  | m  "r-f"r™"i'"t-'' rt 20.00 30.00  p. „,,M „pri iii .,,|iii MM ii. iF ,.,|.n .. ,. , f l  r  f  n  10.00  )  i  |  j  ?  r  [  ,  j  F i g u r e 3 . 2 0 . O n - l i n e L C / M S / M S detection of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I (ion transitions for M R M w e r e : m/z 6 2 4 3 1 9 a n d 6 2 4 -> 4 4 8 ; L C m e t h o d A ) in (A) a q u e o u s solution s p i k e d with the purified metabolites; (B) after 3 0 min of incubation of 2 , 4 - d i e n e V P A - g l u c u r o n i d e with G S H in t h e p r e s e n c e of G S T e n z y m e ; ( C ) after 3 0 m i n of incubation of 2,4-diene V P A - g l u c u r o n i d e with G S H ; (D) after 3 0 m i n of incubation of 2 , 4 - d i e n e V P A - g l u c u r o n i d e only. S i g n a l to n o i s e ratios in (A), (B) a n d (C) a r e ~ 9 0 100, w h e r e a s signal to n o i s e ratio in (D) is 1.  145  In preparation for the G S H addition reaction to 2 , 4 - d i e n e V P A - g l u c u r o n i d e , the G S T activity in the rat liver cytosolic fraction w a s first d e t e r m i n e d u s i n g C D N B a s a s u b s t r a t e (Section 3.4.1).  F o l l o w i n g a 3 0 min incubation of G S H with 2 , 4 - d i e n e V P A -  glucuronide,  V P A - g l u c u r o n i d e I w a s readily  5-GS-3-ene  (Figures 3.20, B a n d C ) .  detected  by  LC/MS/MS  A l t h o u g h n o attempt w a s m a d e to quantitate  absolute  a m o u n t s of the resultant di-conjugate, it w a s estimated, b a s e d o n the intensity of the M S / M S r e s p o n s e s , that the rat liver G S T c a t a l y z e d reaction p r o d u c e d 3.5-fold more product than in the u n c a t a l y z e d buffer reaction.  N o ion s i g n a l c o r r e s p o n d i n g to the  G S H - g l u c u r o n i d e di-conjugate a p p e a r e d for incubations in w h i c h G S H w a s a b s e n t (Figure 3.20, D).  3.5. Preliminary Metabolic Studies of a-Fluorinated VPA Analogues in Rats  3.5.1. Characteristic GC/MS mass spectra of tBDMS derivatives of oc-fluorinated VPA analogues U n d e r E l conditions, the t B D M S derivatives of a-fluoro V P A a n d a - f l u o r o - 4 - e n e V P A a p p e a r e d to p r o d u c e typical [M - 57]+ p e a k s c o r r e s p o n d i n g to the l o s s of a t-butyl group, w h e r e a s the m o l e c u l a r ions w e r e not o b s e r v e d (Figure 3 . 2 1 , A a n d B ) . A n a b u n d a n t fragment ion, m/z 77, w a s found in the G C / M S m a s s s p e c t r a of the T M S a n d t B D M S derivatives of a-fluoro V P A a n d a-fluoro-4-ene V P A (Figure 3 . 2 1 , A and B).  H i g h resolution m a s s s p e c t r a of the derivatives, including the [ H g ] T M S  derivative, confirmed that the m/z  2  77  ion p o s s e s s e d the c h e m i c a l structure [F-  146  S i ( C H 3 ) 2 ] (Table 5), p r e s u m a b l y resulting from the migration of the fluorine ion of the +  h a l o a c i d t B D M S or T M S derivative to the c h a r g e d silicon c e n t e r ( S c h e m e 3.6) (White, E. V . a n d M c C l o s k e y , 1970). S u b s e q u e n t l y , this feature fragment ion m/z 7 7 w a s u s e d in the quantitative  a n a l y s i s of a-fluoro V P A a n d a-fluoro-4-ene V P A in biological  matrices ( S e c t i o n s 2.7). CH  3  CH  tBu  CH  3  ->  m/z S c h e m e 3.6. fluoro V P A .  (M+ - 57)  3  F-Si=CH  m/z  3  77  Formation of the m/z 77 fragment ion from the t B D M S derivative of a -  3.5.2. Metabolism of a-fluorinated VPA analogues in rats T y p i c a l p-oxidation metabolites, n a m e l y ( E ) - 2 - e n e V P A a n d ( E , E ) - 2 , 3 ' - d i e n e V P A w e r e d e t e c t e d in the s e r u m a n d urine of a rat treated with V P A ( F i g u r e 3.22). Similarly, m e t a b o l i s m of 4 - e n e V P A in the rat w a s o b s e r v e d to p r o d u c e ( E ) - 2 , 4 - d i e n e V P A ( s e e S e c t i o n 3.6.3).  Identification of the metabolites w a s b a s e d o n a c o m p a r i s o n  of G C retention times a n d m a s s s p e c t r a with synthetic reference c o m p o u n d s .  Under  similar G C / M S conditions, n o n e of t h e s e metabolites w e r e d e t e c t e d in the s e r u m a n d urine of rats treated with either a-fluoro V P A or a-fluoro-4-ene V P A ( F i g u r e 3.23 a n d S e c t i o n 3.6.3),  indicating a n interruption  in the p-oxidation  analogues.  147  of a-fluorinated V P A  M e t a b o l i s m of a-fluoro V P A in the rat w a s o b s e r v e d to p r o d u c e a - f l u o r o - 4 - e n e V P A a n d a - h y d r o x y V P A , although the a m o u n t s w e r e estimated to b e s m a l l b a s e d on their  respective G C / M S  r e s p o n s e s (Figures 3.23 a n d 3.24).  B e c a u s e the T M S  derivative of a - h y d r o x y V P A p r o d u c e d a better G C c h r o m a t o g r a m a n d g a v e a c l e a n m a s s s p e c t r u m w h i c h w a s identical to that reported ( T a t s u h a r a et a l . , 1987), detection of this metabolite w a s carried out using M S T F A treated urine extracts.  D e s p i t e the  availability of synthetic a-fluoro-3-ene V P A a n d its b r e a k d o w n product 4 - h y d r o x y - 2 - e n e V P A lactone, neither of t h e s e two c o m p o u n d s w a s detected a s a metabolite in the urine of a n a-fluoro V P A treated rat.  Table 5. High resolution mass analysis of the m/z77 fragment ion derived from TMS or tBDMS derivatives of a-fluoro VPA and a-fluoro-4-ene VPA  Compound  C  H  F  Si  [ H ]  Obs. Mass  Calc. Mass  a-F VPA-tBDMS  2  6  1  1  0  77.0227  77.0221  a-F V P A - T M S  2  6  1  1  0  77.0221  77.0221  a-F V P A - [ H ] T M S  2  0  1  1  6  83.0583  83.0599  a-F-4-ene V P A - t B D M S  2  6  1  1  0  77.0215  77.0221  a-F-4-ene V P A - T M S  2  6  1  1  0  77.0223  77.0221  2  6  2  6  a High resolution mass spectra were recorded on either a Kratos MS50 mass spectrometer (70 eV., 150°C) or a Kratos MS80 mass spectrometer interfaced with a Carlo Erba 4160 gas chromatograph (DB1 column: 15 m x 0.32 mm, 0.25 ^im; He: 30 cm/sec; G C : 50°C, 1 min, 50 250°C, 15°C/min).  148  [F-Si(CH ) ]+  A  3  2  lAbundance  COO-tBDMS  5000000  4000000  3000000  [M - 57]H  55 2000000  219  41 97 29  IOOOOOO 4  115 I 20  •>  135  147 176 191 |H1 •|-TTr-i"|vr"i-i"|v , p - i i ry, ,• n , , v, , , , , , , , , , , p , , , •[•, 234 , , , , , , ,26127f ,,,,,, , 40 60 80 100 120' 140 160 180 200 220 240 260 2t  1  B |Abundance 1\1  6000000  COO-tBDMS  5000000 -I  4000000  [M - 57]H 217  41  3000000 -|  95  2000000 57  29 1000000  121. 133 147 _ u l  pl/Z  ->  Figure 3.21.  20  40  60  80  100  120  17 3 189  I '' " 1 "  140  160  1 1  I ' ' I  180  1  1  200  232 45 275 r 1 ' i '' 1 220 240 260 2H 2  -  1  1  1  1  1  1  1  1  G C / M S m a s s s p e c t r a of the t B D M S derivatives of (A) a-fluoro V P A a n d  (B) oc-fluoro-4-ene V P A .  149  COOH  4e+07 3.5e+07 3e + 07 -] 2 . 5e+07 2e + 07 1.5e+07 l e + 07 5000000 0 frime  ' i 12.00 1  ->  1  1  1  ' l 14.00 1  1  1  1  J jU  i • ' ' ' i i i • i | ii i i | 16.00 18J00 20.'00 22'.00  24 ! Oo' 2 6 ! 00  2 8 .' 00  3500000  B 3000000 2500000 -| 2000000  2,3'-diene 4-ene  1500000 2-ene 1000000 -  2,4-diene  4-Hydroxy lactone  50 0000 0 ' fTime  ->  1  1  1  i 11 .00  1  1  1  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 12 . 00 13 .00 14.00 15.00 1 6 . 0 0 1 7 . 0 0 18.00 1 9 . 0 0  F i g u r e 3 . 2 2 (A) G C / M S total ion current chromatogram of the t B D M S derivatives of a n e x t r a c t e d urine s a m p l e from a V P A treated rat. (B) P e a k s for metabolites ( E ) - 2 - e n e V P A , 4 - e n e V P A , 2 , 3 ' - d i e n e V P A , (E)-2,4-diene V P A a n d 4-hydroxy V P A lactone a r e indicated in t h e e x p a n d e d c h r o m a t o g r a m of the lower p a n e l .  150  A  Figure 3.23.  (A)  G C / M S total ion current c h r o m a t o g r a m of the t B D M S d e r i v a t i z e d  extract of a urine s a m p l e from a rat treated with a-fluoro V P A . interest is m a g n i f i e d in the lower p a n e l .  (B)  T h e region of  Neither ( E ) - 2 - e n e V P A nor (E)-2,4-diene V P A  w a s detected.  151  lAbundance 1.2e+0*  ' le + OJ 8e + 07  6e+07  4e+07  COOH  2e + 07  Time  ->  ~r  10.00  (Abundance  1  15 . 00  r  25 . 00  20 . 00  [M - COOTMSF  B  187  40000 35000 30000 25000 147  20000 -  [M - C3H7I+ 15000 -  45  261  10000 5000 -I  [M-15]+ 28  289  115  249  ill! . i .  0 50  100  150  200  250  I i. 300  3 3  |54 350  383  4  400  F i g u r e 3.24. (A) G C / M S total ion current c h r o m a t o g r a m of the T M S d e r i v a t i z e d extract of a urine s a m p l e from a rat treated with a-fluoro V P A . (B) G C / M S m a s s s p e c t r u m of the T M S derivative of the metabolite a-hydroxy V P A . F r a g m e n t a t i o n s a r e identical to the reported d a t a ( T a t s u h a r a et a l . , 1987).  152  [M-57]-* 3^7  14 0 0 0 0 0  CH,CH,CONH I  7  I '  FCCONHCHCOO-1BDMS  1200000  I  CH3CH2CH2 1000000  84 55  800000 4 600000 186 400000  [M-C00tBDMS]-»  41 129  200000  245 158 198  Ll. , fo/Z ->  100  100000  150  200  330  259 I  2  8  1  I  8  3 8  3?4 I' 400  1  250  300  350  441 1  1  1  I 450  73 CH2=OICH  B 80000 -  CHJO^CONHJ  2  FOX)NHC>COC«BDMS I CH3CH2CH2  60000  [M-57]+  40000  345  [M-COOtBDMS]"  1  20000  243259 2 8 5  328  I  1 i 4 1 4 437 LILI.^. tiiJui.iiu^nlii^.jiiLtyi.yt^i.. ,J, ... ^. 9  ,  2  1  3  7  1  R  fr/z ->  Figure 3.25.  300  350  400  G C / M S m a s s s p e c t r a of the t B D M S d e r i v a t i v e s of urinary (A) a-fluoro  V P A - G I n a n d (B) a - f l u o r o - 4 - e n e V P A - G I n .  F r a g m e n t a t i o n s a r e i d e n t i c a l to that of the  synthetic r e f e r e n c e c o m p o u n d s .  153  T w o additional metabolites d e r i v e d from the m e t a b o l i s m of a-fluoro V P A a n d a fluoro-4-ene V P A , respectively, w e r e r e c o g n i z e d b a s e d o n a 2 m a s s unit difference for characteristic p e a k s in the G C / M S m a s s s p e c t r a of their t B D M S derivatives (Figure 3.25). F o l l o w i n g the typical l o s s of a t-butyl group, the putative [M - 57]+ ions a p p e a r e d to match the c o r r e s p o n d i n g t B D M S derivatives of the glutamine c o n j u g a t e s of a-fluoro VPA  (a-fluoro  respectively.  VPA-GIn)  and  a-fluoro-4-ene  S u b s e q u e n t l y , the identities  VPA  (a-fluoro-4-ene  VPA-GIn),  of the metabolites w e r e c o n f i r m e d  by  c o m p a r i n g their G C / M S properties with synthetic reference c o m p o u n d s .  3.6. Comparative Toxicological Studies of 4-Ene VPA and a-Fluoro-4-ene VPA in Rats  3.6.1. Histopathological studies B e c a u s e 4 - P A a n d 4 - e n e V P A are known to i n d u c e m i c r o v e s i c u l a r s t e a t o s i s in rat liver ( K e s t e r s o n et a l . , 1984), t h e s e c o m p o u n d s w e r e s e l e c t e d for this study to s e r v e a s positive s t a n d a r d s . At d o s e s of 1.0 mmol/kg, two rats in the group a d m i n i s t e r e d 4P A w e r e d e a d within the first two d a y s a n d s u b s e q u e n t l y a n additional a n i m a l w a s u s e d for this treatment.  In all other g r o u p s the a n i m a l s s u r v i v e d the 5 d a y treatment.  C h r o n i c administration of either 4 - P A or 4 - e n e V P A to rats resulted in m a s s i v e a c c u m u l a t i o n of lipid in their livers with the w h o l e liver a p p e a r a n c e b e i n g quite similar to that reported previously ( K e s t e r s o n et a l . , 1984).  U p o n s a c r i f i c e , the livers from 4-  P A a n d 4 - e n e V P A treated a n i m a l s w e r e m u c h paler than livers from controls.  154  During  the preparation of liver mitochondria from 4 - P A a n d 4 - e n e V P A treated rats, larger a m o u n t s of a d h e r i n g lipid a s c o m p a r e d to controls w e r e present in the supernatant w h e n the liver h o m o g e n a t e w a s centrifuged. T h e s e two treatment g r o u p s w e r e s c o r e d 4+ for m i c r o v e s i c u l a r s t e a t o s i s a s determined by the light m i c r o s c o p i c e x a m i n a t i o n ( T a b l e 6). Liver s e c t i o n s stained with H & E a n d O i l - R e d - 0 r e v e a l e d that at least 8 5 % of the h e p a t o c y t e s w e r e affected.  T h e p r e s e n c e of lipid w a s diffuse throughout  the  p a n l o b u l a r a r e a a s illustrated in p h o t o m i c r o g r a p h s of the H & E s t a i n e d t i s s u e ( F i g u r e s 3.26, A a n d 3.27, A ) . Of the 3 rats that r e c e i v e d 4 - P A , two of the livers a p p e a r e d to be affected l e s s s e v e r e l y than that of the 4 - e n e V P A treated rats; o n e liver from a 4 - P A treated rat w a s affected at the s a m e level a s 4 - e n e V P A treated rats.  In s o m e livers,  hypertrophy of the Kupffer cells c o u l d be o b s e r v e d . In contrast, the livers from F 2 - 4 - P A a n d a - f l u o r o - 4 - e n e V P A treated rats w e r e similar in a p p e a r a n c e to controls with m u c h l e s s a d h e r i n g lipid present during the preparation of mitochondria. In p r e p a r e d liver s e c t i o n s from treated rats, F 2 - 4 - P A a n d a - f l u o r o - 4 - e n e V P A i n d u c e d lipid d e p o s i t s in l e s s than 2 5 % of the h e p a t o c y t e s ( F i g u r e s 3.26, B a n d 3.27, B, < 2+ for s t e a t o s i s , T a b l e 6).  T h e a s s o c i a t e d lipids w e r e located  only in the periportal a r e a s of the affected livers. O n e half of the livers from rats in the a - f l u o r o - 4 - e n e V P A g r o u p s a n d all livers from the F - 4 - P A group w e r e j u d g e d to be 2  unaffected through the examination of the O i l - R e d - 0 s t a i n e d liver s e c t i o n s (0-1+ for s t e a t o s i s , T a b l e 6).  Inspection of the H & E s t a i n e d liver s e c t i o n s r e v e a l e d a slightly  different result for t h e s e two treatment g r o u p s : all livers from the oc-fluoro-4-ene V P A treated group tested 0-1+ while t h o s e from the F - 4 - P A treated group t e s t e d 2+ (Table 2  155  6).  In a n y c a s e the o b s e r v e d differences in the ability to i n d u c e m i c r o v e s i c u l a r  s t e a t o s i s b e t w e e n 4 - P A a n d F 2 - 4 - P A a n d between 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A w e r e a p p a r e n t a n d significant.  Table 6. Microvesicular steatosis in the livers of rats receiving chronic treatment  3  Microvesicular steatosis  N o . of Treatment  rats  0-1 +  2+  3+  4+  Control  3  3&  0  0  0  (0)  (0)  (0)  0  0  0  3  (0)  (0)  (0)  (3)  4  0  0  0  (0)  (4)  (0)  (0)  0  0  0  4  (0)  (0)  (0)  (4)  2  2  0  0  (4)  (0)  (0)  (0)  (3) 4-PA F -4-PA 2  4-ene V P A a-F-4-ene VPA  a  3 4 4 4  c  The rats were administered i.p. with 4 - P A (1.0 mmol/kg per day), F2-4-PA (1.0 mmol/kg per day), 4-ene V P A (0.7 mmol/kg per day), and oc-fluoro-4-ene V P A (0.7 mmol/kg per day), respectively, for 5 consecutive days, and sacrificed 1 h after their 6th dose.  ^ Microvesicular steatosis was determined by the light microscopic examination of O i l - R e d - 0 stained liver samples. c  The liver samples were stained with hematoxylin-eosin and examined by light microscopy, and the results were presented in the brackets.  156  M i t o s i s w a s o c c a s i o n a l l y o b s e r v e d in the livers from rats d o s e d with either fluorinated or non-fluorinated y o u n g rats.  c o m p o u n d s , a n d is c o n s i d e r e d to be normal  among  V e r y few lipid droplets w e r e detected in the liver s a m p l e s of the control  group (0-1+ for s t e a t o s i s , T a b l e 6) a n d the distribution of the lipid a p p e a r e d at r a n d o m . In all g r o u p s , including t h o s e rats that d e v e l o p e d s e v e r e m i c r o v e s i c u l a r s t e a t o s i s , the a n i m a l s s e e m e d to grow normally a s estimated from their i n c r e a s e d b o d y weights (8 10 g per day).  N o hepatocyte n e c r o s i s c o u l d be clearly e s t a b l i s h e d in a n y of the  treatment g r o u p s . E l e c t r o n m i c r o s c o p i c examination confirmed the p r e s e n c e of s t e a t o s i s in the livers of rats administered 4 - e n e V P A . throughout the c y t o p l a s m .  microvesicular  Lipid droplets w e r e located  M y e l o i d b o d i e s a n d mitochondrial matrix i n c l u s i o n s w e r e  a l s o found in this group (Figure 3.28, A ) . Livers of cc-fluoro-4-ene V P A treated rats, on the other h a n d , exhibited n o n e of t h e s e manifestations. in  liver  tissue  between  the  control  and  d i s t i n g u i s h e d (Figure 3.28, B).  157  N o ultrastructural  oc-fluoro-4-ene  V P A groups  differences could  be  Figure 3.26. P h o t o m i c r o g r a p h s of liver sections from rats administered 4 - e n e V P A (A) a n d a-fluoro-4-ene V P A (B), respectively, at 0.70 mmol/kg daily for 5 d a y s . M i c r o v e s i c u l a r steatosis w a s diffuse throughout the liver lobule in 4 - e n e V P A treated a n i m a l s , a s e v i d e n c e d by the density of fat droplets that a p p e a r a s tiny white spots, but w a s a b s e n t in tx-fluoro-4-ene V P A treated rats (H & E, x 450). P: portal v e i n ; C : central vein.  158  Figure 3.27. P h o t o m i c r o g r a p h s of liver s e c t i o n s from rats treated with 4 - P A (A) a n d F 2 4 - P A (B), respectively, at 1.00 mmol/kg daily for 5 d a y s . M i c r o v e s i c u l a r s t e a t o s i s w a s diffuse throughout the liver lobule in 4 - P A treated a n i m a l s , a s e v i d e n c e d by the density of fat droplets that a p p e a r a s tiny white spots, but w a s a b s e n t in F 2 - 4 - P A treated rats (H & E , x 450). P : portal v e i n ; C : central v e i n .  159  F i g u r e 3.28.  E l e c t r o n m i c r o g r a p h s of h e p a t o c y t e s from rats a d m i n i s t e r e d 4 - e n e V P A  (A) a n d a - f l u o r o - 4 - e n e V P A (B), respectively, at 0.70 m m o l / k g daily for 5 d a y s . N u m e r o u s lipid v a c u o l e s (L), myeloid b o d i e s (M), a n d mitochondrial matrix inclusions (I) c a n be s e e n in 4 - e n e V P A treated animals (x 1320) but not in a - f l u o r o - 4 - e n e V P A treated rats (x 1120).  160  3.6.2.  GSH status in the liver of 4-ene VPA and a-fluoro-4-ene VPA treated  animals T h e hepatic mitochondria p r e p a r e d from all treatment g r o u p s w e r e found to b e intact.  T h e r e w a s n o o b v i o u s inhibition of oxidative p h o s p h o r y l a t i o n in a n y of the  mitochondrial preparations b a s e d o n m e a s u r e d respiratory control i n d e x e s of 5.1 to 12.0 a n d A D P / O ratios of 2 . 3 to 3.1 ( P e d e r s e n et a l . , 1978).  T h e activity of the  c y t o s o l i c m a r k e r e n z y m e , lactate d e h y d r o g e n a s e , in the mitochondrial  preparations  w a s 0 . 5 1 % of that in the h o m o g e n a t e s , indicating a negligible amount of cytosolic contamination in the mitochondrial preparations. T h e recycling a s s a y for m e a s u r i n g total G S H ( G S H + G S S H ) w a s s u g g e s t e d to be a s e n s i t i v e a n d specific e n z y m a t i c p r o c e d u r e ( A n d e r s o n , 1985). coefficients  of i n t e r - a s s a y variation  determination  were  estimated  In o u r h a n d s , the  to b e 9 . 1 % a n d 8 . 2 % for  of G S H at the levels of 2 nmol a n d 5 n m o l , respectively (n = 4).  S u b s e q u e n t l y , G S H levels in whole liver h o m o g e n a t e s a n d in m i t o c h o n d r i a  were  m e a s u r e d at 0 . 7 5 , 2 , a n d 4 h after the rats were administered 1.41 m m o l / k g of either 4 e n e V P A or a-fluoro-4-ene V P A . Control h o m o g e n a t e a n d mitochondrial G S H v a l u e s w e r e d e t e r m i n e d to b e 3 3 . 6 nmol/mg protein a n d 3.71 n m o l / m g protein, respectively, a n d w e r e c o m p a r a b l e to p u b l i s h e d v a l u e s (Tirmenstein a n d N e l s o n , 1989).  Liver  h o m o g e n a t e G S H levels at 4 h w e r e d e p l e t e d by 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A to 5 6 % a n d 7 2 % of control, respectively (Table 7). In the c a s e of 4 - e n e V P A , t h e G S H level in the h o m o g e n a t e r e a c h e d its lowest point at 2 h a n d w a s relatively constant without r e c o v e r y during t h e s u b s e q u e n t 2 h period (Table 7). H e p a t i c mitochondrial  161  G S H l e v e l s r e m a i n e d u n c h a n g e d in rats d o s e d with a - f l u o r o - 4 - e n e V P A ( T a b l e 8), while 4 - e n e V P A treatment r e d u c e d mitochondrial G S H to 6 8 % of control (Table 8). T h i s d e c r e a s e of mitochondrial G S H by 4 - e n e V P A w a s d e t e c t e d at 2 h p o s t - d o s e a n d r e m a i n e d that w a y during the following 2 h (Table 8). After 2 4 h of fasting, the 5th d a y control levels of hepatic G S H in the c h r o n i c treatment group d e c r e a s e d to 23.2 nmol/mg protein (Table 7), most likely d u e to a n e n h a n c e d rate of G S H efflux from the liver to other t i s s u e s (Vogt a n d R i c h i e , 1993).  In  contrast to the results of the acute treatment, neither 4 - e n e V P A nor a-fluoro-4-ene V P A s h o w e d a n y effect on the total hepatic G S H content following six daily d o s e s of 0.70 m m o l / k g of either drug (Table 7). After 5 d a y s , mitochondrial G S H w a s e l e v a t e d in 4 - e n e V P A treated rats to twice that of control, but r e m a i n e d the s a m e a s the control in rats d o s e d with oc-fluoro-4-ene V P A (Table 8). A n inhibition of mitochondrial G R activity w a s d e t e c t e d in the 4 - e n e V P A treated group of rats, the e n z y m e activity b e i n g r e d u c e d to 4 3 % of control. N o significant d e c r e a s e in mitochondrial G R activity w a s o b s e r v e d in rats a d m i n i s t e r e d a-fluoro-4-ene V P A (Table 8).  162  c  CD  E c 'CD •*—'  o 1  CL D)  CD +-» O C  CO CO LO  o  +1 o o  CM CO C\J  CO CD +1 CO 00 CO CO  it  +1  CO  CVJ  ZZ-  o I-  O CO  s  E c  cvi cvT  CD co CO CD +  +1  CD  CO  cts c  •4—' CO  E  CO  CD  CO Q. 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JZ  0  TJ  0 l_  •tf ,_  2 O 3  CD C  CO co CL X  -r E 0 a ^ O  0  TJ  £2  c  o o  > i CZ « 0 0 •<t _0 ci  I ©  +i £  CO  2 75)  c CO  0  E  CD 03£ CD "O 5  CO 2 CO  0  _  0  I  o E  TJ  =s.  0 3  I00  0  _Q  CO CO  s  4 0  E Q CO TJ CO 0 5 C  >^'E  §  o 0 CL X  0  v  Q.O  .  w 92 CO 2  8 co "§<  51 i<  iS 0 cb £  8  ?T  E 2  J2  T3 -D  9J  > 0 O 0  0  0 <5 jo i  SCCOO CO Cr  CO TJ  8 2 2 8  E o  »- TJ CO  CD 0 co  TJ  c  CO  0  s  E -S  co  2  > Q  TJ  CO  o d  lCO lco _3  > 0  CO JZ  2 £  Z §  0 £ i — JZ 0  co  3= TJ  •° 1o o a)  T= <D C CO CO JO  2° COcoP ^  *: 0 C TJ CO CO  JZ  CL  C O  >,"a  co E  TJ  3.6.3.  Differences of 4-ene VPA and a-fluoro-4-ene VPA in producing (E)-2,4-  diene VPA and the NAC conjugate of (E)-2,4-diene VPA in chronically treated rats Urinary a n d s e r u m 4 - e n e V P A , (E)-2,4-diene V P A a n d a-fluoro-4-ene V P A w e r e d e t e r m i n e d u s i n g t h e a s s a y d e s c r i b e d in the S e c t i o n 2.9.4 a n d S e c t i o n 3.7.5. T h e metabolite (E)-2,4-diene V P A w a s detected in the urine a n d s e r u m of rats treated with 4 - e n e V P A for 5 d a y s (Figure 3.29, A ) . G l u c u r o n i d e s of 4 - e n e V P A a n d (E)-2,4-diene V P A in urine w e r e estimated from the differences in t h e free a c i d  concentrations  d e t e r m i n e d before a n d after alkaline hydrolysis. T h u s , urinary recovery of 4 - e n e V P A w a s 5.9 ± 1.5 (imol a s the free a c i d a n d 18.5 ± 1.3 |imol a s t h e g l u c u r o n i d e .  Urinary  (E)-2,4-diene V P A a c c o u n t e d for ~ 2 . 6 % of the 5th d o s e of 4 - e n e V P A , b e i n g 0.61 ± 0.07 (imol a s the free a c i d a n d 2.1 ± 0.3 (imol a s t h e g l u c u r o n i d e .  U n d e r similar  G C / M S conditions, neither urine nor s e r u m s a m p l e s from rats a d m i n i s t e r e d a-fluoro-4e n e V P A for 5 d a y s w e r e found to contain a detectable amount of (E)-2,4-diene V P A (Figure 3.29, B ) . T h e differences in urinary oc-fluoro-4-ene  V P A before a n d after  alkaline hydrolysis w e r e minimal (44.4 ± 4.4 (imol vs. 4 6 . 9 ± 3.1 u.mol), s u g g e s t i n g that negligible a m o u n t s of the glucuronide w e r e formed. T h e urinary r e c o v e r y of oc-fluoro-4e n e V P A during d a y 5 w a s ~ 3 7 % of the d o s e administered o n that day.  165  Se*07 -  prime  ->  12.00  14.00  16.00  18.00  20.00  22.00  24.00  26.00  28.00  B 4e+07  3e + 07  4  2e + 07  4  le+07  I ' " I' 1' I ' ' ' I ' 1^' • • i • • • • i • • • • i • • • • t • • • • i • ' 1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 18.00 20.00 2 2 . 0 0 2 4 . 0 0 2 6 . 0 0 2 8 . 0 0  -T—I—I—I—I  (Time ->  1  1  r|  1  1  F i g u r e 3 . 2 9 . G C / M S total ion current c h r o m a t o g r a m s of t B D M S d e r i v a t i v e s of extracted urine s a m p l e s from (A) 4 - e n e V P A a n d (B) a-fluoro-4-ene V P A treated rats.  166  C H  1  3  Abundance  7  CH2-CH=CH-CH-COOSi(CH3)2-C(CH ) 3  3  S-CH -CH-COOSi(CH )2-C(CH3)3 2  140000 -j  3  HNOCCH3  "1  1 120000-j  1  [NACtBDMS]"  100OO0-;  1  [M-tBDMS]"  1  60000 -J  I 40000-]  1 1  [M-15]+  20000-  m  516  Jiik  L  200  300  400  r - •! 1  Mass/Charge  Figure 3.30.  (A) G C / M S m a s s spectrum of t B D M S derivative of t h e urinary N A C  c o n j u g a t e of ( E ) - 2 , 4 - d i e n e V P A in 4 - e n e V P A treated rats, w h i c h is identical to that of the s y n t h e s i z e d r e f e r e n c e c o m p o u n d .  167  Ion 471.00 ociu (roo 7V0201002.O Hun Bt 03: 44 PH PSI on Hod Nov 24.  1993  Ion 415.00 amu from 7V0201002.0  tounflancL'  Ion 276.00 amu from 7V0201002.d  250O000-  B  Ion 474.00 aau from 7V0901009.d Run  a t 09: 13 PM PST on Ned Nov 24.  1993  Ion 415.00 aau f r o a 7V0901009.d  Abundance  Ion 276.00 aau f r o a 7V0901009.d  T  1  '  1~  21.5  F i g u r e 3 . 3 0 {cont'd).  (B) T h e N A C conjugate of ( E ) - 2 , 4 - d i e n e V P A (retention  time:  2 0 . 1 7 min) f o u n d in t h e urine of 4 - e n e V P A treated rats (C) but not in t h e urine of a f l u o r o - 4 - e n e V P A treated rats.  T h r e e fragment ions, m/z 4 7 4 , m/z 4 1 5 a n d m/z 2 7 6  w e r e u s e d in t h e identification of the conjugate.  168  T h e 4 - e n e V P A metabolite (E)-2,4-diene V P A c a n b e biotransformed to its G S H conjugate in the liver a n d s u b s e q u e n t l y through the mercapturate p a t h w a y b e e x c r e t e d in urine a s the N A C conjugate ( K a s s a h u n et a l . , 1991). T h e N A C conjugate of (E)-2,4d i e n e V P A w a s detected in all urine s a m p l e s from 4 - e n e V P A treated rats ( F i g u r e s 3.30, A a n d B) but not in the urine of rats d o s e d with a - f l u o r o - 4 - e n e V P A (Figure 3.30, C ) , further confirming that the m e t a b o l i s m of ot-fluoro-4-ene V P A in rats d o e s not l e a d to the formation of (E)-2,4-diene V P A .  3.7. Comparative Pharmacokinetic and Metabolic Studies of 4-Ene VPA and a Fluoro-4-ene VPA in Rats  3.7.1.  LC/MS/MS characterization of the NAC conjugate of 2-fluoro-4,5-  epoxypentanoic acid in the urine of rats treated with cc-fluoro-4-ene VPA T h e M S / M S d a u g h t e r ion s p e c t r u m of the putative urinary N A C conjugate of a fluoro-4-ene V P A a p p e a r e d to r e s e m b l e that of the non-fluorinated counterpart ( S e c t i o n 3.3.3).  S i m i l a r to 5 - N A C - 4 - h y d r o x y V P A lactone, the neutral l o s s of k e t e n e from the  protonated  5 - N A C - 2 - p r o p y l - 2 - f l u o r o - 4 - h y d r o x y V P A lactone  was a  p r o c e s s , resulting in the daughter ion at m/z 2 8 0 (Figure 3.31).  predominant  T h e prominent  d a u g h t e r ion at m/z 162 w a s likely to a r i s e from the neutral loss of 2-fluoro-4-hydroxy V P A lactone. T h e neutral loss of 2-fluoro-5-thiol-4-hydroxy V P A lactone w o u l d result in the d a u g h t e r ion at m/z 130, a n d the c o m b i n e d loss of k e t e n e , c a r b o n m o n o x i d e a n d a  169  m o l e c u l e of water g a v e fragment m/z 2 3 4 (Figure 3.31).  A n identical M S / M S C I D  fragmentation pattern w a s o b s e r v e d for the synthetic reference c o m p o u n d .  O-C + 2H _280 i  CH COiHN 3  F  S 62  COOH MH 322  +  100  4x3  F i g u r e 3 . 3 1 . M S / M S m a s s spectrum of t h e urinary metabolite 2-propyl-2-fluoro-5-(A/a c e t y l c y s t e i n - S - y l ) - 4 - h y d r o x y p e n t a n o i c a c i d lactone in rats treated with oc-fluoro-4-ene V P A at 1.4 m m o l / k g . F r a g m e n t a t i o n s for the metabolite, w h i c h a r e identical to t h e synthetic reference c o m p o u n d , a r e d i s c u s s e d in the text. T h e configuration of the c o m p o u n d r e m a i n s to b e clarified.  170  3.7.2. LC/MS/MS characterization of the glutamine conjugate of a-fluoro-4-ene VPA in the bile and urine of rats treated with a-fluoro-4-ene VPA A n initial L C / M S / M S s c r e e n for the biliary a n d urinary metabolites of a-fluoro-4e n e V P A directed o u r attention to the ion m/z 2 8 9 w h i c h a p p e a r e d to match the m o l e c u l a r weight of protonated a-fluoro-4-ene V P A - G I n .  T h e M S / M S C I D s p e c t r u m of  this putative glutamine conjugate w a s c h a r a c t e r i z e d by s c i s s i o n of t h e a - a m i n o group on t h e glutamine moiety with c h a r g e retention b e i n g p o s s i b l e o n either s i d e of t h e molecule.  T h u s , t h e neutral loss of 2-fluoro-2-propyl-4-pentenamide p r o d u c e d a  prominent d a u g h t e r ion at m/z 130, w h e r e a s t h e fragment registered at m/z 1 6 0 w a s indicative of t h e formation of protonated 2-fluoro-2-propyl-4-pentenamide (Figure 3.32). T h e neutral l o s s of /V-formyl glutamine w o u l d result in the d a u g h t e r ion at m/z 115 a n d , following a further loss of h y d r o g e n fluoride, p r o d u c e the fragment at m/z 9 5 (Figure 3.32).  T h e s e a s s i g n m e n t s w e r e s u p p o r t e d by the similar fragmentation  pattern  o b s e r v e d for a-fluoro V P A - G I n (Section 3.8.1), w h e r e the d a u g h t e r ions a s s o c i a t e d with the saturated V P A moiety w e r e 2 m a s s units higher than t h o s e s e e n in t h e M S / M S C I D of a - f l u o r o - 4 - e n e V P A . A n identical M S / M S C I D fragmentation pattern w a s o b s e r v e d for the synthetic reference c o m p o u n d .  171  + 2 H , 160—,  130  COOH CONH  2  MH 100i  289  0/ _ /o  130 84 95  1151  160 J  100  150  200  250  \ m/z 300  F i g u r e 3 . 3 2 . M S / M S m a s s s p e c t r u m of the urinary metabolite A^-(2-fluoro-2-propyl-4pentenoyl)glutamine in rats treated with a - f l u o r o - 4 - e n e V P A at 1.4 m m o l / k g . F r a g m e n t a t i o n s for t h e metabolite, w h i c h a r e identical to t h e synthetic reference c o m p o u n d , are d i s c u s s e d in the text. T h e configuration of the c o m p o u n d r e m a i n s to b e clarified.  172  3.7.3. Quantitation of the NAC conjugates in the urine of rats treated with either a -fluoro-4-ene VPA or 4-ene VPA M e t a b o l i s m of 4 - e n e V P A in rats p r o d u c e d two N A C c o n j u g a t e s , 5 - N A C - 4 hydroxy V P A lactone a n d 5 - N A C - 3 - e n e V P A .  Quantitation  of t h e s e  conjugated  metabolites w a s carried out u s i n g the a s s a y d e s c r i b e d in the S e c t i o n s 2.5.7 a n d 3.3.6. T h e urinary 5 - N A C - 4 - h y d r o x y V P A lactone a n d 5 - N A C - 3 - e n e V P A w e r e d e t e r m i n e d to be 2 . 1 % a n d 0.6%, respectively, of the 4 - e n e V P A d o s e (Table 9).  U n d e r similar  c o n d i t i o n s , the N A C conjugate of (E)-2,4-diene V P A , n a m e l y 5 - G S - 3 - e n e V P A , c o u l d not be d e t e c t e d by L C / M S / M S in the urine s a m p l e s c o l l e c t e d from rats treated with a fluoro-4-ene V P A at 1.4 mmol/kg, w h i c h w a s in a g r e e m e n t with our results o b t a i n e d earlier for the G C / M S a s s a y (Section 3.6.3). Quantitatively, the lactone N A C conjugate from oc-fluoro-4-ene V P A w a s estimated to be only 0 . 0 5 % of the d o s e (Table 9).  3.7.4. Quantitation of the glutamine conjugate in the bile and urine of tx-fluoro-4ene VPA treated rats C a l i b r a t i o n c u r v e s for L C / M S / M S quantitation of biliary a n d urinary a-fluoro-4e n e V P A - G I n w e r e linear o v e r the r a n g e s tested with the coefficients of determination greater than 0.99.  T h e coefficients of inter-day variation w e r e 3 . 9 % a n d 5 . 9 % at the  c o n c e n t r a t i o n s of 6 4 u.g/mL a n d 4 u.g/mL, respectively (n = 3), a n d the coefficients of intra-day variation w e r e 3 . 1 % a n d 1.0% at the c o r r e s p o n d i n g c o n c e n t r a t i o n s (n = 3). Quantitative  a n a l y s i s of cc-fluoro-4-ene V P A - G I n  indicated that the  urinary  glutamine conjugate c o n s i s t e d of 2 2 % of the d o s e (Table 9). In a s e p a r a t e experiment,  173  the excretion of biliary a-fluoro-4-ene V P A - G I n w a s d e t e r m i n e d to b e 1.1 ± 0.2 (imol during a 6 h p e r i o d , a c c o u n t i n g for ~ 0 . 7 % of the d o s e .  D e s p i t e t h e availability of a  synthetic reference c o m p o u n d which w o u l d facilitate the L C / M S / M S s c r e e n of the metabolite, the glutamine conjugate of 4 - e n e V P A w a s not d e t e c t e d in either the urine or bile s a m p l e s c o l l e c t e d from the rats administered 4 - e n e V P A .  Table 9. Urinary Phase II metabolites of 4-ene VPA and tx-fluoro-4-ene VPA Parent D r u g  Glucuronides^ ([imol)  L-Glutamine conjugates ((imol)  NAC Conjugates (limol)  4-Ene V P A  13.4 ± 6 . 5 * 10.5 ± 4 . 8 * *  N.D.  9.6 ± 1.5  oc-F-4-ene V P A  N.D.  67.0 ± 36.5  0.19 ± 0 . 0 6  3  a Rats were dosed i.p. with either 4-ene VPA or cc-fluoro-4-ene VPA at 1.4 mmol/kg. Urine samples were collected over a 24 h period. The metabolite levels were expressed as mean ± S.D. (n = 4). N.D.: not detectable. b The glucuronides were estimated from the difference between the hydrolyzed and unhydrolyzed samples. * Data obtained from the alkaline hydrolysis (60°C, pH 12.5 and 60 min). ** Data obtained from the enzymatic hydrolysis (400 units of p-glucuronidase/500 |iL of urine, 37°C, pH 5.7 and 18 h). c The metabolism of 4-ene VPA in rats resulted in 5-NAC-3-ene VPA (2.1 + 0.19 (imol) and 5-NAC-4-hydroxy VPA lactone (7.5 ± 1.5 (imol). The data shown here represents the summation of the two urinary NAC conjugates. Only one NAC conjugate, 5-NAC-2-fluoro-4-hydroxy VPA lactone, was detected in the urine of rats treated with oc-fluoro-4-ene VPA.  174  0  3.7.5.  Urinary recovery of 4-ene VPA and cc-fluoro-4-ene VPA Quantitation of s e r u m or urinary 4 - e n e V P A a n d a-fluoro-4-ene V P A w a s carried  out u s i n g a G C / M S p r o c e d u r e d e v e l o p e d earlier in this laboratory ( Y u et a l . , 1995). our h a n d s , calibration c u r v e s w e r e linear o v e r the r a n g e s t e s t e d .  In  T h e inter-assay  variation w e r e l e s s than 1 0 % for either urine or s e r u m s a m p l e s (Table 10).  Table 10. Inter-assay variations for determination of serum and urinary 4-ene VPA or oc-fluoro-4-ene VPA by GC/MS Compounds  3  Serum  Urine  A s s a y 1, A s s a y 2  A s s a y 1, A s s a y 2  4-ene V P A  1 A8 ,  a-F-4-ene V P A  2.73,  b  1.60 2.77  1.58,  1.53  2.57,  2.64  a Control serum or urine samples spiked with 4-ene VPA or cc-fluoro-4-ene VPA at 8 |ig/mL. were assayed by selective ion monitoring ion: m/z 199 for 4-ene V P A and m/z 77 for cc-fluoro-4-ene VPA (Yu et al., 1995). b The numbers represent the GC/MS peak area ratios using octanoic acid as an internal standard  T h e intact oc-fluoro-4-ene V P A excreted into the urine w a s d e t e r m i n e d to b e 135.9 ± 1 1 . 8 u m o l , w h i c h w a s ~ 10-fold the 10.7 ± 2.0 (imol estimated for 4 - e n e V P A . U p o n hydrolysis, urinary 4 - e n e V P A w a s i n c r e a s e d twice a s m u c h a s that in the u n h y d r o l y z e d s a m p l e s , s u g g e s t i n g a n e x t e n s i v e glucuronidation of 4 - e n e V P A (Table 9). C o n s i s t e n t with a n earlier report (Dickinson et a l . , 1984), a l k a l i n e h y d r o l y s i s of the urine s a m p l e s resulted in a higher estimation of the glucuronidation of 4 - e n e V P A than the (3-glucuronidase c a t a l y z e d hydrolysis (Table 9). M e t a b o l i s m of a - f l u o r o - 4 - e n e V P A  175  in rats, o n t h e other h a n d , p r o d u c e d a negligible amount  of t h e c o r r e s p o n d i n g  g l u c u r o n i d e (135.9 ± 11.8 jimol v s . 1 3 0 . 6 ± 1 3 . 7 umol following t h e e n z y m a t i c hydrolysis).  T h e urinary  (E)-2,4-diene V P A in the 4 - e n e V P A treated  d e t e r m i n e d to b e 2.6 ± 0.5 urnol with 3 1 % in the g l u c u r o n i d e form.  rats w a s  C o l l e c t i v e l y , the  urinary r e c o v e r i e s of 4 - e n e V P A a n d a-fluoro-4-ene V P A , including their metabolites, w e r e 10.4 a n d 5 8 % of the d o s e s , respectively.  S u c h a low recovery of 4 - e n e V P A in  the urine is most likely d u e to the extensive metabolism of this c o m p o u n d (Rettenmeier et a l . , 1 9 8 6 ; S i n g h et a l . , 1988).  3.7.6. Liver uptake and protein binding properties of 4-ene VPA and a-fluoro-4ene VPA At d o s e s of 1.4 mmol/kg, rat liver uptake of 4 - e n e V P A a n d oc-fluoro-4-ene V P A a p p e a r e d to b e e q u a l at 1 h post d o s e , being 0.96 ± 0.11 a n d 0.89 ± 0 . 1 9 u.mol/g of wet liver, respectively.  C o n s i d e r i n g that the weight of the rat livers h a d a m e a n v a l u e of  11.8 ± 1.1 g , disposition of 4 - e n e V P A a n d its a-fluorinated a n a l o g u e in liver t i s s u e w a s e s t i m a t e d to b e ~ 3 % of the d o s e at 1 h post d o s e . In vitro, parallel i n c r e a s e s in the s e r u m free drug l e v e l s of 4 - e n e V P A a n d a fluoro-4-ene V P A w e r e apparent within the s e r u m concentration r a n g e s of 0.25 to 2 . 9 0 urnol/ml_ (Figure 3.33). T h e binding to s e r u m proteins w a s in the r a n g e of 4 0 - 6 0 % at the lower drug c o n c e n t r a t i o n s a n d d e c r e a s e d to ~ 2 0 % at t h e higher c o n c e n t r a t i o n s .  176  o  0.0  0.5  1.0  1.5  2.0  2.5  3.0  Total concentration (|jmol/ml_)  F i g u r e 3.33. R e l a t i o n s h i p between the free a n d total c o n c e n t r a t i o n s of 4 - e n e V P A (O) or oc-fluoro-4-ene V P A (V) in rat s e r u m . Protein binding w a s a s s e s s e d v i a ultrafiltration of 4 5 0 u l of the s e r u m s a m p l e s s p i k e d with the s o d i u m salt of either 4 - e n e V P A or a f l u o r o - 4 - e n e V P A u s i n g a n A m i c o n micropartition s y s t e m . E a c h v a l u e r e p r e s e n t s the m e a n of two m e a s u r e m e n t s of duplicates.  177  3.7.7.  Serum drug concentration-time profiles and pharmacokinetic modeling for  4-ene VPA and a-fluoro-4-ene VPA dosed to rats After a d o s e of 1.4 mmol/kg i.p. to rats of either 4 - e n e V P A or a - f l u o r o - 4 - e n e V P A , the s e r u m concentration-time profiles c o n s i s t e d of a brief a b s o r p t i o n p h a s e , followed by a rapid elimination p h a s e during a period of 3 0 to 180 min ( F i g u r e s 3.34 a n d 3.35). T h e apparent elimination rate constant estimated from the linear portion of the c u r v e for 4 - e n e V P A w a s greater than that of oc-fluoro-4-ene V P A ( T a b l e 11). W h i l e the s e r u m concentration of oc-fluoro-4-ene V P A c o n t i n u e d to d e c l i n e , a s e c o n d s e r u m drug c o n c e n t r a t i o n p e a k w a s evident for the 4 - e n e V P A treated rats, o c c u r r i n g at ~ 3 0 0 min ( F i g u r e s 3.34 a n d 3.35). T h e apparent total c l e a r a n c e s of 4 - e n e V P A a n d a-fluoro4 - e n e V P A , estimated a s d o s e / A U C , a p p e a r e d to be c o m p a r a b l e (Table 11). T h e metabolite (E)-2,4-diene V P A w a s detected only in the s e r u m of 4 - e n e V P A treated rats, r e a c h i n g its p e a k concentration at 6 0 min post d o s e (Figure 3.34). S i m i l a r to the parent d r u g , a s e c o n d s e r u m p e a k concentration w a s a l s o o b s e r v e d for the d i e n e metabolite, w h i c h c o i n c i d e d with the s e c o n d p e a k of 4 - e n e V P A at ~ 3 0 0 min. T h e s e r u m concentration-time c u r v e for rats receiving 4 - e n e V P A c o u l d be fitted by the p r o p o s e d two compartment p h a r m a c o k i n e t i c m o d e l (Figure 3.34). T h e time-lag T1 w a s arbitrarily c h o s e n a s 2 0 0 min for the best-fit. T h e c a l c u l a t e d v a l u e s of microrate c o n s t a n t s , k , k , k a  1 0  1 2  a n d k -|, w e r e 0.048 ± 0 . 0 0 5 , 0 . 0 2 7 ± 0 . 0 0 5 , 0 . 0 0 7 ± 0.001 2  a n d 0.004 ± 0.001 m i n , respectively. A s i n g l e compartment m o d e l w a s u s e d to fit the - 1  s e r u m concentration-time c u r v e for rats treated with oc-fluoro-4-ene V P A (Figure 3.35).  178  T h e micro-rate c o n s t a n t s estimated from the m o d e l w e r e 0.078 ± 0.016 m i n  - 1  for k  a  and  0 . 0 1 8 + 0.002 m i n ' for k i . 1  0  0  100  200  300  400  500  600  Time (min)  F i g u r e 3.34. S e r u m concentration-time profiles of 4 - e n e V P A (O) a n d ( E ) - 2 , 4 - d i e n e V P A (A) in the rats d o s e d i.p. with 4 - e n e V P A at 1.4 m m o l / k g . E a c h v a l u e w a s the m e a n concentration o b t a i n e d from four a n i m a l s . T h e s o l i d line r e p r e s e n t s the best-fit for s e r u m 4 - e n e V P A levels u s i n g the two compartment o p e n m o d e l with first-order a b s o r p t i o n , first-order elimination a n d a time-lag.  179  0  100  200  300  400  500  600  Time (min)  F i g u r e 3.35. Drug concentration-time profile of a-fluoro-4-ene V P A in rat s e r u m after a n i.p. injection at 1.4 mmol/kg. E a c h v a l u e w a s the m e a n concentration o b t a i n e d from four a n i m a l s . T h e solid line represents the best-fit u s i n g the o n e compartment o p e n m o d e l with first-order absorption a n d elimination.  180  Table 11. Apparent pharmacokinetic parameters of 4-ene VPA and a-fluoro-4-ene VPA in rats Parameter  4-Ene V P A  C m a x (umol/mL)  2.34 ± 0.44  2.58 ± 0 . 1 5  0.0259 ± 0 . 0 0 3 5  0.0173 ± 0 . 0 0 0 7  27.1 ± 3 . 7  40.2 ± 1.3  194.8 ± 2 7 . 5  206.1 ± 16.6  7.29 ± 1.00  6.83 ± 0.55  K  (min" )  0  (min)  0  1  t-i/2 AUC  (|imol-min/ml_)  CLjp ( m l _ / m i n k g )  c  c  a-F-4-ene V P A  a  a  a Rats were dosed i.p. with either 4-ene V P A or cc-fluoro-4-ene V P A at 1.4 mmol/kg. The data were expressed as mean ± S.D. (n = 4). b The terminal elimination rate constants (K) were estimated from the slopes of linear portion of the serum drug concentration-time profiles and the half-life (1*7/2) calculated as 0.693/K. c The areas under curves (AUC) were estimated using the trapezoidal method and the apparent clearance (CLjp) calculated as dose/AUC.  3.8. Comparative Pharmacokinetic, Pharmacodynamic and Metabolic Studies of VPA and cc-Fluoro VPA in Mice  3.8.1. Structural characterization and quantitation of the glutamine conjugate of a-fluoro VPA A preliminary metabolic study c o n d u c t e d in rats r e v e a l e d that a-fluorinated analogues  c o u l d undergo conjugation with  L-glutamine  ( S e c t i o n 3.5.2).  In  VPA this  investigation, a n L C / M S / M S s c r e e n for metabolites in the urine of m i c e treated with a fluoro VPA  directed our attention to the ion m/z 291 w h i c h a p p e a r e d to match the  181  m o l e c u l a r weight of protonated a-fluoro V P A - G I n .  T h e M S / M S C I D of the  putative  glutamine conjugate w a s c h a r a c t e r i z e d by s c i s s i o n of the a - a m i n o g r o u p o n  the  glutamine moiety with c h a r g e retention p o s s i b l y b e i n g on either s i d e of the m o l e c u l e . T h u s , the neutral l o s s of 2-fluoro-2-propylpentanamide p r o d u c e d a prominent d a u g h t e r ion at m/z 130, w h e r e a s the fragment registered at m/z 162 w a s indicative of the formation of protonated 2-fluoro-2-propylpentanamide (Figure 3.36).  T h e neutral l o s s  of A/-formyl glutamine w o u l d result in the daughter ion at m/z 115 a n d , following a further l o s s of h y d r o g e n fluoride, p r o d u c e the fragment at m/z 9 5 (Figure 3.36).  An  identical M S / M S C I D fragmentation pattern w a s o b s e r v e d for the synthetic reference compound. The  GC/MS  methods  d e s c r i b e d earlier for  detection  of  a-fluoro  VPA-GIn  (Section 3.5.2) w a s not u s e d for quantitation b e c a u s e it w a s s u s p e c t e d that, like Np h e n y l a c e t y l g l u t a m i n e (Kamerling et a l . , 1979), a-fluoro V P A - G I n w a s thermally labile. In this respect, L C / M S / M S u s i n g electrospray a p p e a r e d to b e s u p e r i o r in the s e n s e of a v o i d i n g the u s e of high temperature.  Calibration c u r v e s w e r e o b s e r v e d to b e linear  o v e r the r a n g e s tested with the coefficients of determination greater than 0.99. coefficients  of  inter-day  variation  were  estimated to  be 8 . 3 % a n d 6 . 4 %  The  at  the  c o n c e n t r a t i o n s of 3 7 . 0 u.g/mL a n d 2.3 u.g/mL, respectively (n = 3). T h e coefficients of intra-day variation w e r e 6 . 4 % a n d 6 . 9 % at the c o r r e s p o n d i n g c o n c e n t r a t i o n s (n = 3). A s u b s e q u e n t L C / M S / M S quantitative a n a l y s i s of a-fluoro V P A - G I n indicated that the urinary glutamine conjugate c o n s i s t e d of 3 3 . 3 % of the d o s e ( T a b l e 13).  O n the  other h a n d , the glutamine conjugate of V P A w a s not d e t e c t e d in urine s a m p l e s  182  c o l l e c t e d from m i c e administered V P A , d e s p i t e the availability of a synthetic reference c o m p o u n d w h i c h w o u l d facilitate the detection of the metabolite.  + 2H, 162—  130  COOH CONH  2  MH 100i  291  0/ _ /o  130 84 162 100  150  200  250  \ m/z 300  F i g u r e 3 . 3 6 . M S / M S m a s s s p e c t r u m of the urinary metabolite /V -(2-fluoro-2propylpentanoyl)glutamine in m i c e treated with a-fluoro V P A at 0 . 8 3 mmol/kg. F r a g m e n t a t i o n s w h i c h a r e identical to the synthetic reference c o m p o u n d a r e d i s c u s s e d in t h e text. T h e configuration of the c o m p o u n d r e m a i n s to b e clarified. 2  183  3.8.2. Detection and pharmacokinetic characterization of V P A and a-fluoro V P A in the mouse brain, serum and urine Quantitation of V P A a n d a-fluoro V P A in the brain, s e r u m or urine w a s carried out u s i n g a G C / M S a s s a y d e v e l o p e d earlier in this laboratory ( Y u et a l . , 1995).  In o u r  h a n d s , calibration c u r v e s s h o w e d g o o d linearity with the coefficients of determination greater than 0.99. T h e inter-assay variation w e r e l e s s than 1 0 % (Table 12).  Table 12. Inter-assay variations for determination of V P A or a-fluoro V P A in the brain, serum or urine by GC/MS Compounds  3  Brain  Serum  Urine  A s s a y 1, A s s a y 2  A s s a y 1, A s s a y 2  A s s a y 1, A s s a y 2  VPA  2.21°,  2.22  2.14,  2.09  2.16,  2.06  a-Fluoro V P A  1.77,  1.76  1.73,  1.87  1.54,  1.57  a Control brain, serum or urine samples spiked with V P A or a-fluoro V P A at 8 ng/ml_ were assayed by selective ion monitoring ion: m/z 201 for V P A and m/z 77 for a-fluoro V P A (Yu et al., 1995). b The numbers represent the G C / M S peak area ratios using octanoic acid as an internal standard.  184  CD CD  |1  E to .=» E  Q  Q  CO CO  ^ 8^ CD  _  -£  o  "So ^  O p +1 +1 CO o d  CM  o E  LU  CO  d +1 cq O  0  CO C  CO  CO CD  E CO CO  CO CD  W CO  => O  —  Q  _  E  q o +1 +1 +1 CO 1^  h-  o d  oO 0  co co CD L_  CL  in c\i 1  CD CD  CD CL  CD C  CL  4—»  CO  ' l _  CM  _Q  o  CD  0  CO CD C  2.2  I  £  CD O  ^  * § CO Z5  o d  d +1 +1 +1 1 _  s_  CO  o d  CO CM  C o  CL  E o O  < CL >  << CL CL > > CD O c 0 CM  LZT  0  O ' 15  o  •  CO  J£ ^  JD CO  © Q. E CO  o E E  0 E 0  CO CO CO  *  JZ CL  I- >  TD  0 0  0  O  0 3=  ~o  0  CO CO  E o CO  < CL > CVJ  LlT  o CO CO  T3  -t—•  0  o  S-o  CO >< 0 JZ T3 C C =  1= 3 * ZI "0  CO O  o 3 -»—* i0  i_  c o T3 0 -*—'  O C  2>£  0 c  2? 0  0  CO "D 0 0  co  0  CO  0 c 0  CO  5  Z3 0  CO E CO CO  CO  «= CO  \  CO  E  o  > CO  g  T3 0 0  T3 0 0 CL  CL  0  CD  o 0  CO CO  0  CO  O O  >  Q  T3  O  CO  CO  •a 0 0 > o o 0 l_  D "D T3 0  E  CO  X  CD  O  O  D L_  CD  o  0  CO  T>  c  0  c 0 o c o o  c  to 'c 'E T3  CO  CO  +1  1  0  o  Q CO  CO CD C  CL >  .Q  o t -  <  c 0 0  O  C  "  CO o  0  Q  < CL >  CD c  +1  o c  O Zi  0  .E  -a  5 £  0  "5 JO  co 0 E 0  co  CO JZ Q.  0  JZ  I— CO  CO  185  T h e fragment ion m/z 77 that w a s confirmed to h a v e the structure [ F - S i ( C H 3 ) 2 ]  +  (Section 3.5.1) w a s u s e d in G C / M S S I M m o d e for the quantitation of a-fluoro V P A a n d the c h o i c e of this ion p r o v e d to be more sensitive than the typical [M - 57]+ ion at m/z 2 1 9 a n d e q u a l l y reliable. F o r e x a m p l e , at 6 0 min following a d o s e of 0.83 m m o l / k g , the brain concentration of a-fluoro V P A determined by S I M with ion m/z77  w a s 0.37 + 0.03  u,mol/g of t i s s u e a n d 0.37 ± 0.02 umol/g of t i s s u e by S I M with ion m/z 2 1 9 . At 6 0 min after a higher d o s e of 2.08 mmol/kg, the concentration w a s d e t e r m i n e d to b e 0.99 ± 0.08 (imol/g of t i s s u e by S I M with ion m/z 77, consistent with the v a l u e of 0.97 ± 0.08 u. mol/g of t i s s u e by S I M with ion m/z 2 1 9 . a - F l u o r o V P A w a s c h a r a c t e r i z e d by a s l o w a c c e s s into the m o u s e brain e v e n though  the  serum  concentration  of  the  drug  increased  rapidly  following  the  administration (Figure 3.37, A ) . After d o s e s of 0.83 m m o l / k g , a-fluoro V P A r e a c h e d its p e a k concentration in the s e r u m b e t w e e n 15 a n d 3 0 min w h e r e a s the highest brain drug c o n c e n t r a t i o n did not o c c u r until 4 5 min.  S i m u l t a n e o u s c u r v e fitting u s i n g a two  compartment m o d e l g a v e absorption rate c o n s t a n t s of 0.25 + 0.03 m i n compartment a n d 8.50 ± 1.70 (x 1 0 ) m i n - 4  - 1  - 1  for the brain compartment.  for the s e r u m T h e apparent  efflux of a-fluoro V P A from the brain mirrored its s l o w elimination from the s e r u m (Figure 3.37, A ) , w h e r e a s the micro-rate c o n s t a n t s c a l c u l a t e d for the d r u g to l e a v e the brain a n d the circulation w e r e 0.10 ± 0.02 m i n  - 1  a n d 1.50 ± 0.20 (x 1 0 ) - 3  min , - 1  respectively, the former being far greater. T h e p e a k concentration of V P A in the brain a n d s e r u m a p p e a r e d to o c c u r within 15 min p o s t d o s e a s no absorption p h a s e w a s o b s e r v e d (Figure 3.37, B). Elimination of  186  V P A from the brain a n d s e r u m w a s rapid (Figure 3.37, B), h a v i n g a p p a r e n t c o n s t a n t s of 2.58 ± 0.51 (x 1 0 " ) m i n 2  - 1  rate  a n d 2.02 ± 0.13 (x 1 0 " ) m i n ' , respectively. 2  1  T h e p e a k brain concentration of a-fluoro V P A w a s e s t i m a t e d to o c c u r at 4 5 min after a d o s e of 0.83 m m o l / k g , being 0.411 ± 0.041 umol/g of t i s s u e a n d r e m a i n e d at this level for at least another 3 5 min.  At the s a m e d o s e , the highest brain concentration  d e t e r m i n e d for V P A w a s 0.472 ± 0.048 umol/g of t i s s u e at 15 min p o s t d o s e .  In  contrast, the p e a k s e r u m level of a-fluoro V P A (45 min p o s t d o s e ) w a s higher than the highest concentration o b s e r v e d for V P A (15 min p o s t d o s e ) , b e i n g 4.44 ± 0 . 6 8 u.mol/ml_ vs. 2 . 8 8 ± 0.20 urnol/mL at 0.83 mmol/kg d o s e s . E x p r e s s e d a s the brain-to-serum drug c o n c e n t r a t i o n ratio, a-fluoro V P A h a d a v a l u e of 0.093 d e t e r m i n e d at the time of the p e a k brain concentration w h i c h r e m a i n e d constant for the following 5.5 h, w h e r e a s V P A h a d a ratio of 0.16 e s t i m a t e d at 15 min p o s t d o s e . T h e primary metabolite of V P A in the p-oxidation pathway, ( E ) - 2 - e n e V P A , w a s found in the s e r u m a n d urine of V P A treated mice but w a s not detected in mice a d m i n i s t e r e d a-fluoro V P A under similar G C / M S conditions. V P A underwent e x t e n s i v e g l u c u r o n i d a t i o n , 4 9 . 4 % of the total drug excreted in the urine b e i n g c o n j u g a t e d .  Only  6 . 5 % of a-fluoro V P A w a s estimated to be in its g l u c u r o n i d e e s t e r in the urine (Table 13).  C o l l e c t i v e l y , the urinary r e c o v e r i e s of V P A a n d a-fluoro V P A , including their  metabolites, w e r e 5 7 . 8 a n d 8 4 . 7 % of the d o s e s , respectively (Table 13).  187  A  1000  10  0  100  200  300  400  Time (min)  F i g u r e 3.37. (A) Drug concentration-time profiles of a-fluoro V P A in the m o u s e brain a n d s e r u m after a n i.p. d o s e of 0.83 mmol/kg. • - a - F l u o r o V P A in the s e r u m (u.g/ml_); O - a-fluoro V P A in the brain (|ig/g of tissue). E a c h v a l u e r e p r e s e n t s a n a v e r a g e of 4 a n i m a l s . T h e solid lines represent the p h a r m a c o k i n e t i c m o d e l curve-fitting.  188  B  1000  c o  100  CO i_  •*—>  c CD o c o o  CD 13  10  0  50  100  150  Time (min)  F i g u r e 3.37 (cont'd). (B) Drug concentration-time profiles of V P A in the m o u s e brain a n d s e r u m a n d its metabolite ( E ) - 2 - e n e V P A in the s e r u m after a n i.p. d o s e of V P A of 0.83 m m o l / k g . T - V P A in the s e r u m (u.g/ml_); V - V P A in the brain (u.g/g of t i s s u e ) ; A ( E ) - 2 - e n e V P A in the s e r u m (u.g/mL). E a c h v a l u e represents a n a v e r a g e of 4 a n i m a l s .  189  3.8.3.  Anticonvulsant activity and elevation of the synaptosomal GABA levels V P A anticonvulsant testing in m i c e w a s performed by injecting P T Z s . c . at 10  min after the drug d o s e followed by a 3 0 min o b s e r v a t i o n period ( S w i n y a r d et a l . , 1989). T h e E D 5 0 w a s determined to b e 0.83 m m o l / k g , in a g r e e m e n t with the literature v a l u e of 0.83 - 1.04 m m o l / k g ( S w i n y a r d et a l . , 1 9 8 9 ; L o s c h e r et a l . , 1991) ( F i g u r e 3.38). T h e a n t i c o n v u l s a n t activity of a-fluoro V P A w a s e v a l u a t e d by giving P T Z at 4 5 min after the drug d o s e , the time point at w h i c h the drug r e a c h e d its a p p a r e n t p e a k c o n c e n t r a t i o n in the brain. T h e E D 5 0 e s t i m a t e d for a-fluoro V P A w a s 1.70 m m o l / k g (Figure 3.38). T h e brain s y n a p t o s o m a l G A B A level in control m i c e w a s d e t e r m i n e d to b e 19.68 ± 0 . 7 5 n m o l / m g of protein by the G C / M S method.  T h i s v a l u e is c o n s i s t e n t with  p u b l i s h e d d a t a of 16.6 ± 4 . 2 3 nmol/mg of protein o b t a i n e d by a r a d i o r e c e p t o r a s s a y ( L o s c h e r et a l . , 1981). At d o s e s of 0.83 mmol/kg a n d 2.08 m m o l / k g of V P A , the G A B A l e v e l s w e r e e l e v a t e d to 110 a n d 1 2 1 % of control at 15 min p o s t d o s e a n d w e r e i n c r e a s e d to 118 a n d 1 2 7 % of control at 3 0 min p o s t d o s e d e s p i t e a d e c r e a s e in brain drug c o n c e n t r a t i o n (Table 14).  S i n c e P T Z i n d u c e d s e i z u r e s g e n e r a l l y started 5 to 7  min after s . c . injection of the c o n v u l s a n t agent, the onset of the protective effect of V P A is temporally correlated with the elevation of s y n a p t o s o m a l G A B A l e v e l s . A n i n c r e a s e in the s y n a p t o s o m a l G A B A levels w a s a l s o o b s e r v e d in a-fluoro V P A treated m i c e a n d the d e g r e e of elevation w a s similar to that i n d u c e d by V P A , b e i n g 119 a n d 1 2 5 % of control at 4 5 min following administration of the drug at 0.83 m m o l / k g a n d 2.08 m m o l / k g , respectively (Table 14). U n l i k e the V P A c a s e , w h e r e - 5 0 % of the a n i m a l s e x p o s e d to P T Z w e r e protected by the drug at a d o s e of 0 . 8 3 m m o l / k g , a  190  -fluoro  V P A s h o w e d little anticonvulsant effect  at the s a m e d o s a g e d e s p i t e  the  o b s e r v e d elevation of G A B A . T h e brain drug concentration-time profile of a-fluoro V P A r e a c h e d a plateau during 2 5 to 6 0 min after a d o s e of 2.08 m m o l / k g , the protection of m i c e against P T Z i n d u c e d s e i z u r e s p r e s e n t e d a s t e a d y i n c r e a s e until 4 5 min, w h e n a significant elevation of G A B A w a s o b s e r v e d (Table 15).  191  Drug doses (mmol/kg) F i g u r e 3.38. E v a l u a t i o n of the anticonvulsant activities for V P A (V) a n d its a fluorinated a n a l o g u e (O). T h e s o d i u m salt of V P A or a-fluoro V P A in a q u e o u s solution w a s a d m i n i s t e r e d i.p. to m i c e at different concentrations. P T Z (85 mg/kg) w a s given s . c . at 15 min ( V P A ) or 4 5 min (a-fluoro V P A ) a n d the a n i m a l s w e r e o b s e r v e d for a n additional 3 0 min for s e i z u r e s which w e r e rated a s : 0, no s e i z u r e ; 0.5, c l o n i c s e i z u r e , a n i m a l r e m a i n e d o n its feet; 1, tonic-clonic s e i z u r e , a n i m a l lost its b a l a n c e ( S w i n y a r d et al. 1 9 8 9 ; L o s c h e r a n d Vetter, 1985). S e i z u r e s o c c u r r e d at a m e a n time of 7.2 ± 4 . 3 min in control m i c e following the d o s e of P T Z .  192  at  c  B  o  < 5  S  "~ d d C M + +1 +i +1 I  co o  I  3  1^ * * * I : o> cp CVJ  o  1  ^ C  c  o o  CO T -  i -  LO  cvj cvj cvj  E  < >  CO CO  co D TO M—  O  c o  d  CO CO  d  00 00 o o  cvi cvi  II  CO  O)  <  M—  CQ o  * i —  G  d ^ ^ ^ +1 +1 +1 +1  < cn C\l N CD E cvi co o  CO  CVJ  CM  C\l  4  CVJ  LO CVJ  E  0  CO  0 $ _co — 0 > 0  < CD « E o co o 4—»  CL CO  co  zs o  i _  CL >  CO  ° Q  o F  E  oo  CO CO  CO 00  CO  00  d  d  cvi cvi  4—»  c o  CO CD  C  .i E  I— —  LO T -  O CO  LO T -  o CO  CL  0  E ico  193  4—»  0  TO  Zi  CO J0Z  CVJ  «d  "§ +i c CO L O  o  c  ZI  o  CO >  — TO  C O °  co E  ±CO CO  E  ^  i  0  .£  CO J Z  s se s co  o  CO O CO 4.1 LCO II CO O  CO  Zi  TO O CO  CO  co  TJ  X 0  ?Z  O CD  +  L  d  c  CM "—' 0 Q> JO  .E  « 2 «-  JO  73 >T3 0 0  0  2 .|  0 £ CO 4=  -2 JZ  0  TO CO CO  cc  co  I .c  -^;£ §  0  §  CO  5  CO  CO  .E £ 'E E o C  O  0 0  TO  0 0  JO  c 0  co  _co  CO  CO  .E  O  < > c o  CO  —  0  CO  0  Q. o. CD 0  o  o  Q.  0  0  0  JZ  CD  o  CO  Z)  o E  <  CO  d 1?  0  +1 c E  O CD  cr  JO CO  •  LO O  CO  CO  c  CO UJ  CD  c  CO  c  (0  c "E  0  0 i _  * * cvi co  LO  II  c o 4—« ZJ o  "O  < c "a> o *  CD  c  4—» "4—  TJ  LO "3"  CO  LO  CO  CD  o LO o co •<* CO  JO  CO 4-»  0  CO OO  E  CO  TO CO  CD =* to -7:  0  i  'c 'E  CD  CO  < o CD cvj  > CO  0 0 CO CO D  0 55  TO O - -  9> w  5  <  0  CO O  _  2 O O  < T-°O  < COE  0  5 sa E 0  ^  o  CO  0  4—•  CO  CD c  0  'CO  i_  JO  0  $ 0  0  O  > CD ° o c -2 0-  52  m ® 2  jE £  ~ CO  Q) >  CL CO  c  CO  0  CD  Table 15. Relationship between protection against PTZ-induced seizures, synaptosomal GABA levels, and brain a-fluoro VPA concentrations % Protection  Brain concentration (nmol/g of t i s s u e )  Synaptosomal G A B A % increase  25(25)  0.98 (25 - 30)  4.0 (25)  4 4 (35)  0.97 (30 - 45)  9.0* (30)  6 3 (45)  0.97 (45 - 60)  2 5 . 5 * (45)  6 3 (60)  1.12(60-80)  13.0* (60)  5 6 (120)  1.20 (120)  17.4* (120)  9  0  0  a Percentage of animals protected against PTZ induced seizures. PTZ (85 mg/kg) was given by s.c. injection at the time points indicated in the brackets following an i.p. dose of a-fluoro VPA at 2.08 mmol/kg (n = 8). b A second set of animals served to measure the brain levels of a-fluoro VPA following a dose of 2.08 mmol/kg. The drug levels were expressed as the average of two concentrations determined at two time points (min) indicated in the brackets (n = 5 at each time point). c A third set of animals served for measuring synaptosomal GABA levels which were determined at 2 5 - 1 2 0 min following the dose of a-fluoro VPA at 2.08 mmol/kg. The values were expressed relative to controls with the time points indicated within the brackets (min, n = 5). The GABA levels of controls were determined to 20.01 ± 0.75 and 19.35 ± 0.72 nmol/mg of protein at 15 and 120 min, respectively, following an i.p. dose of normal saline (n = 5 at each time point). * The increase was statistically significant (Student's t-test was conducted on the determined synaptosomal GABA concentrations, p < 0.05. Estimates of the variance were made based on the within mean squares derived from the ANOVA (Bolton, 1990)).  194  4. Discussion  4.1. Metabolic Formation and Degradation of the GSH Conjugate of (E)-2,4-Diene VPA  4.1.1. Bioactivation of (E)-2,4-diene VPA via glucuronidation The  biotransformation  of V P A to its metabolites entails two major p h a s e I  p a t h w a y s , of w h i c h the s e c o n d a r y metabolite (E)-2,4-diene V P A m a y e m e r g e from either pathway, n a m e l y the m i c r o s o m a l P 4 5 0 c a t a l y z e d d e h y d r o g e n a t i o n of ( E ) - 2 - e n e VPA  ( K a s s a h u n a n d Baillie, 1993) or the mitochondrial p-oxidation of 4 - e n e V P A  (Baillie a n d Rettenmeier, 1989). ( E ) - 2 , 4 - D i e n e V P A h a s b e e n s h o w n to b e hepatotoxic in rats ( K e s t e r s o n et a l . , 1984) a n d is s u s p e c t e d to play a n important role in V P A i n d u c e d liver injury ( K a s s a h u n et a l . , 1 9 9 4 ; S e c t i o n 3.6). In this study, L C / M S / M S a n d N M R d a t a w e r e u s e d to clearly identify two n e w G S H - r e l a t e d metabolites of (E)-2,4d i e n e V P A , n a m e l y the G S H - g l u c u r o n i d e di-conjugates of (E)-2,4-diene V P A , in the bile of rats d o s e d with (E)-2,4-diene V P A .  Sufficient on-line L C / M S / M S d a t a w a s a l s o  o b t a i n e d to indicate the p r e s e n c e of the N A C - g l u c u r o n i d e di-conjugate of ( E ) - 2 , 4 - d i e n e V P A in both rat bile a n d urine.  T h e detection of the N A C - g l u c u r o n i d e di-conjugate in  bile is in a c c o r d a n c e with our o b s e r v a t i o n s of formyl a m p h e t a m i n e m e t a b o l i s m in rats, w h e r e b y all metabolites of the G S H conjugates a l o n g the mercapturic a c i d pathway c o u l d b e identified in the bile (Borel a n d Abbott, 1995).  195  The  d e t e c t e d G S H - g l u c u r o n i d e di-conjugates fall mainly into two c a t e g o r i e s  a c c o r d i n g to w h e t h e r the conjugate is p - g l u c u r o n i d a s e sensitive ( G S H - g l u c u r o n i d e I) or p - g l u c u r o n i d a s e resistant ( G S H - g l u c u r o n i d e II).  T h e p - g l u c u r o n i d a s e resistant form of  the di-conjugate p r e s u m a b l y results from a n o n - e n z y m a t i c intramolecular migration of the primary 1-O-acyl-glucuronide ( S p a h n - L a n g g u t h a n d B e n e t , 1992). T h i s a s s i g n m e n t of structure w a s b a s e d on earlier findings that V P A g l u c u r o n i d e c a n u n d e r g o p H d e p e n d e n t rearrangement a n d the resulting isomeric c o n j u g a t e s a p p e a r resistant to c l e a v a g e by p - g l u c u r o n i d a s e ( D i c k i n s o n et a l . , 1984).  Theoretically, the  migration  c o u l d o c c u r sequentially to form four positional i s o m e r s , n a m e l y 1-, 2-, 3- a n d 4 - O - a c y l g l u c u r o n i d e s ( S c h e m e 4.1) either in vivo or in vitro after the bile w a s c o l l e c t e d . It is not c l e a r to w h i c h position the migration took p l a c e , a n d 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II w a s likely a mixture of 2-, 3- a n d 4 - O - a c y l g l u c u r o n i d e s .  COOH O ^ I ^ ^ O H  HO\ COOH  ( 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II)  ( 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I)  S c h e m e 4 . 1 . P r o p o s e d a c y l migration of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e I to 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II.  196  Differences b e t w e e n t h e s e two t y p e s of a c y l g l u c u r o n i d e s of the identified dic o n j u g a t e s w e r e not limited to their sensitivity towards (3-glucuronidase but a l s o to their M S / M S C I D properties.  T h e latter w a s reflected in the full d a u g h t e r ion s p e c t r u m  o b t a i n e d for 5 - G S - 3 - e n e V P A - g l u c u r o n i d e II w h e r e b y s c i s s i o n of the l i n k a g e b e t w e e n the a c y l moiety a n d glucuronic a c i d w a s not a favored p r o c e s s , s u g g e s t i n g improved strength of the C - 0 ester b o n d after migration.  T h e d a t a o b t a i n e d in this study  i n d i c a t e s that the normally u s e d neutral l o s s s c a n of 176 D a to detect g l u c u r o n i d e s by L C / M S / M S (Straub et a l . , 1987) w o u l d not be suitable to s e a r c h for the i s o m e r i z e d 5G S - 3 - e n e V P A - g l u c u r o n i d e II.  W h e t h e r this difference in fragmentation r e p r e s e n t s a  g e n e r a l p h e n o m e n o n for positional a c y l g l u c u r o n i d e s r e m a i n s to be investigated. T h e d i s c o v e r y of the G S H - g l u c u r o n i d e di-conjugates of ( E ) - 2 , 4 - d i e n e V P A w a s to our mind significant in that the g l u c u r o n i d e ester of this V P A metabolite a p p e a r s to represent a major bioactivated form of this hepatotoxic d i e n e .  It h a s b e e n p r o p o s e d  that the d o u b l e b o n d s of (E)-2,4-diene V P A are activated v i a the formation of the c o r r e s p o n d i n g C o A thioester in either mitochondria (from 4 - e n e V P A ) or e n d o p l a s m i c reticulum (from ( E ) - 2 - e n e V P A ) a n d thereby a d d to the terminal position of the d i e n e through a M i c h a e l addition reaction ( K a s s a h u n et a l . , 1 9 9 1 ; 1994).  H o w e v e r , direct  e x p e r i m e n t a l e v i d e n c e h a s not b e e n reported to pin-point the role of C o A in the conjugation reaction of (E)-2,4-diene V P A with G S H . U s i n g A/-acetylcysteamine a s a structural mimic of C o A , a n in vitro investigation r e v e a l e d that this thioester of (E)-2,4d i e n e V P A c o u l d react s p o n t a n e o u s l y with G S H a n d the conjugation reaction w a s greatly e n h a n c e d by rat hepatic G S T e n z y m e s (Section 3.4).  197  N o reaction c o u l d be  d e t e c t e d w h e n the d i e n e free a c i d w a s u s e d a s a substrate (Section 3.4).  T h i s result  w a s in a g r e e m e n t with findings obtained from c h e m i c a l s y n t h e s i s , i.e., (E)-2,4-diene V P A d o e s not react with G S H u n l e s s first b e i n g c o n v e r t e d to its e s t e r form ( K a s s a h u n et a l . , 1991). In this study, the in vitro incubation of G S H with 2 , 4 - d i e n e V P A - g l u c u r o n i d e led to the formation of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e , a n d this conjugation reaction w a s further  c a t a l y z e d by  rat  liver  G S T enzyme.  These  results, together  with  the  c h a r a c t e r i z a t i o n of the G S H - g l u c u r o n i d e di-conjugate formed in vivo in (E)-2,4-diene V P A treated rats, w e r e the first e v i d e n c e that G S H conjugation of ( E ) - 2 , 4 - d i e n e V P A w a s not d e p e n d e n t o n the C o A thioester, i.e. the g l u c u r o n i d e w o u l d s e r v e e q u a l l y to activate the resulting unsaturated ester to react with G S H . T h u s , G S " thiolate arising from the G S T promoted deprotonation of G S H (Armstrong, 1991) attacks the activated terminal d o u b l e b o n d of 2,4-diene V P A - g l u c u r o n i d e to form the G S H - g l u c u r o n i d e diconjugate v i a either 5,6- or 1,6-addition with the latter b e i n g the dominant p r o c e s s ( S c h e m e 4.2). probably  T h e conjugation reaction of G S H with the unsaturated g l u c u r o n i d e is  limited  to  the  cytosolic  compartment  because ^  uridine  5'-  d i p h o s p h o g l u c u r o n o s y l t r a n s f e r a s e , the e n z y m e c a t a l y z i n g g l u c u r o n i d a t i o n , is l o c a l i z e d in the m e m b r a n e of the e n d o p l a s m i c reticulum (Mulder et a l . , 1990).  F o l l o w i n g the  mercapturic a c i d b i o s y n t h e s i s pathway, the G S H - g l u c u r o n i d e s of ( E ) - 2 , 4 - d i e n e V P A are d e g r a d e d to the c o r r e s p o n d i n g N A C - g l u c u r o n i d e s a n d are eventually e x c r e t e d in the urine.  198  It is l e s s likely that the G S H - g l u c u r o n i d e di-conjugate w a s formed from 5 - G S - 3 e n e V P A , b e c a u s e the di-conjugate in bile w a s determined to be 7-fold that of the c o r r e s p o n d i n g G S H mono-conjugate following a d o s e of ( E ) - 2 , 4 - d i e n e V P A to rats.  If  the p r e c u r s o r of the di-conjugate w a s i n d e e d 5 - G S - 3 - e n e V P A , glucuronidation of the G S H c o n j u g a t e w o u l d n e e d to be very efficient. A high rate of glucuronidation is known to d e m a n d sufficient lipid solubility of the substrate (Mulder et a l . , 1990), a n d 5 - G S - 3 e n e V P A d o e s not a p p e a r to fulfill this requirement. A d d i t i o n a l e v i d e n c e to support the h y p o t h e s i s that the G S H - g l u c u r o n i d e d i conjugate is formed from the glucuronide ester is the fact that 2 , 4 - d i e n e V P A g l u c u r o n i d e a p p e a r s to be the most a b u n d a n t metabolite following a d o s e of (E)-2,4d i e n e V P A to the rat.  F o r e x a m p l e , the 2 , 4 - d i e n e V P A - g l u c u r o n i d e u s e d in this study  w a s readily isolated from the bile of rats treated with the d i e n e . M e t a b o l i c profiling in patients r e c e i v i n g V P A therapy r e v e a l e d that the amount of c o n j u g a t e d ( E ) - 2 , 4 - d i e n e V P A in the urine w a s 7.1-fold that of the unconjugated d i e n e metabolite, indicating e x t e n s i v e glucuronidation of (E)-2,4-diene V P A in vivo in the h u m a n ( K a s s a h u n et a l . , 1990).  T h u s , a significant quantity of the unsaturated g l u c u r o n i d e is a v a i l a b l e for  conjugation with G S H . T h e hepatotoxic metabolite (E)-2,4-diene V P A c a n a l s o be f o r m e d v i a the P 4 5 0 c a t a l y z e d d e h y d r o g e n a t i o n of (E)-2-ene V P A ( K a s s a h u n a n d Baillie, 1993) with levels of the d i e n e b e i n g s e v e r a l fold that p r o d u c e d during the m e t a b o l i s m of V P A in rats ( L o s c h e r et a l . , 1992) a s well a s in h u m a n m i c r o s o m a l i n c u b a t i o n s ( F a b r e et a l . , 1992). A recent quantitative a n a l y s i s c o m p a r i n g biotransformation of ( E ) - 2 - e n e V P A a n d 4 - e n e  199  V P A in rats found that the G S H conjugate of (E)-2,4-diene V P A f o r m e d from ( E ) - 2 - e n e V P A w a s l e s s than that from 4 - e n e V P A ( K a s s a h u n et a l . , 1994). It w a s s u g g e s t e d that metabolic  activation  of  (E)-2-ene  V P A through  the  CoA  mediated  pathway  in  e n d o p l a s m i c reticulum w a s not a s efficient a s 4 - e n e V P A bioactivation in mitochondria ( K a s s a h u n et a l . , 1994).  H o w e v e r , our detection of the G S H - g l u c u r o n i d e di-conjugate  of ( E ) - 2 , 4 - d i e n e V P A implies that a major portion of the (E)-2,4-diene V P A d e r i v e d from ( E ) - 2 - e n e V P A metabolism c o u l d in fact react with hepatic G S H v i a the g l u c u r o n i d e dependent process.  Further investigations into the toxicological s i g n i f i c a n c e of this  p r o c e s s with regards to the relative hepatotoxicity of ( E ) - 2 - e n e V P A a n d V P A ( L o s c h e r et a l . , 1993) n e e d to be e x a m i n e d . Glucuronidation  represents  a major  p h a s e II metabolic  pathway  for  those  c o m p o u n d s that c a n afford to l o s e a reactive proton, s u c h a s a l c o h o l s , a m i n e s a n d c a r b o x y l i c a c i d s ( F a e d , 1984).  Conjugation of x e n o b i o t i c s with D-glucuronic a c i d may  take p l a c e directly or following introduction of a n appropriate functional group into the parent m o l e c u l e upon p h a s e I metabolism.  A l t h o u g h it is g e n e r a l l y a c c e p t e d that  reaction of x e n o b i o t i c s with D-glucuronic a c i d l e a d s to c o n j u g a t e s that a r e  rapidly  e x c r e t e d b e c a u s e of their hydrophilic nature, a n i n c r e a s i n g body of e v i d e n c e s u g g e s t s that d r u g s c a n be bioactivated through the glucuronidation pathway to bind with G S H ( S h o r e et a l . , 1995) or with proteins ( Z i a - A m i r h o s s e i n i et a l . , 1994).  A m o n g these  g l u c u r o n i d e s , the a c y l g l u c u r o n i d e s are by far the most reactive ( Z i a - A m i r h o s s e i n i et al., 1994). F o r e x a m p l e , the incubation of V P A - g l u c u r o n i d e with h u m a n s e r u m albumin w a s d e m o n s t r a t e d to g e n e r a t e covalent V P A - p r o t e i n a d d u c t s (Williams et a l . , 1992).  200  T h e g l u c u r o n i d e m e d i a t e d reaction of x e n o b i o t i c s with G S H or proteins m a y follow a transacylation m e c h a n i s m , w h e r e the reactive c e n t e r is the a c y l c a r b o x y l c a r b o n a n d the glucuronic a c i d moiety is d i s p l a c e d by free thiols, t y r o s i n e or lysine r e s i d u e s in the target proteins ( F a e d , 1984).  A n e x a m p l e of s u c h a reaction is the  formation of clofibric a c i d G S H thioester that o c c u r r e d w h e n 1-O-clofibryl g l u c u r o n i d e w a s m i x e d with G S H . T r a n s a c y l a t i o n w a s s u g g e s t e d to be the m e c h a n i s m ( S h o r e et al., 1995). Alternatively, the c a r b o h y d r a t e ring of the g l u c u r o n i d e m a y o p e n to give a s u g a r c h a i n h a v i n g a n a l d e h y d e functional group. T h e a l d e h y d e m a y then attack a free a m i n o group o n proteins to form a n imine Schiff b a s e w h i c h w o u l d ultimately rearrange to a 1-amino-2-keto protein adduct (Ding et a l . , 1993).  Tolmetin glucuronide was  o b s e r v e d to covalently bind to h u m a n s e r u m albumin v i a this imine m e c h a n i s m (Ding et al., 1993). T o our k n o w l e d g e , the conjugation of 2 , 4 - d i e n e V P A - g l u c u r o n i d e with G S H is the first r e c o r d e d i n s t a n c e in which a drug is activated by glucuronidation to react with G S H by M i c h a e l addition. T h e c o n s e q u e n c e s of forming this metabolite with respect to V P A a s s o c i a t e d hepatotoxicity are far from clear, although o n e w o u l d s u s p e c t that this is o n e more reaction that w o u l d contribute to the o b s e r v e d d e c r e a s e in h e p a t i c G S H reported for V P A ( J e z e q u e l , A . M . et a l . , 1984; C o t a r i u et a l . , 1990).  T h e 2,4-diene  V P A is hepatotoxic in the rat ( K e s t e r s o n et a l . , 1984) a n d the g l u c u r o n i d e e s t e r is the main form of this metabolite found in the urine of rats ( S e c t i o n 3.6.3) a n d patients on V P A therapy ( K a s s a h u n et a l . , 1990).  G S H d e f i c i e n c y c a u s e d by the c h a l l e n g e of  reactive c o m p o u n d s is known to result in cell injury a n d death d u e largely to the  201  impaired anti-oxidant function of the G S H redox s y s t e m ( D e L e v e a n d K a p l o w i t z , 1991). In v i e w of idiosyncratic nature of V P A hepatotoxicity, a n y metabolite of the drug that reacts with G S H s h o u l d be c o n s i d e r e d s u s p e c t , particularly in patients w h o , b e c a u s e of genetic a n d / o r environmental factors, may be a l r e a d y c o m p r o m i s e d with respect to GSH. T h e toxicological s i g n i f i c a n c e of 2,4-diene V P A - g l u c u r o n i d e formation h a s yet to be a s s e s s e d .  Normally the liver would be the logical target o r g a n , b e i n g the site for  reactive metabolite formation, but other t i s s u e s might a l s o b e affected, s u c h a s the kidney, in w h i c h g l u c u r o n i d e s are e x c r e t e d .  A l t h o u g h a rare o c c u r r e n c e , V P A is  reported to c a u s e renal toxicity c h a r a c t e r i z e d by m a r k e d a c c u m u l a t i o n of lipids in the proximal tubular epithelium  a n d alterations  in mitochondria ( H a w k i n s a n d  Brewer,  1993). T h e c o m m o n excretion of the glucuronide of (E)-2,4-diene V P A in patients on V P A therapy, however, is e v i d e n c e that formation of a reactive metabolite d o e s not n e c e s s a r i l y predict toxic c o n s e q u e n c e s .  202  203  4.1.2. Intrahepatic degradation of the GSH conjugates formed during 4-ene VPA metabolism The microsomal  hepatotoxic metabolite 4 - e n e V P A h a s b e e n s h o w n to a r i s e from the P 4 5 0 c a t a l y z e d desaturation of V P A (Rettie  et a l . , 1987).  Further  biotransformations of 4 - e n e V P A include the m i c r o s o m a l P 4 5 0 m e d i a t e d e p o x i d a t i o n of the terminal d o u b l e b o n d a n d mitochondrial p-oxidation (Prickett a n d B a i l l i e , 1986). T h e resultant 4 , 5 - e p o x y V P A in e n d o p l a s m i c reticulum a n d ( E ) - 2 , 4 - d i e n e V P A - C o A in m i t o c h o n d r i a react with the residing G S H in e a c h s u b c e l l u l a r compartment ( K a s s a h u n et a l . , 1994), p r e s u m a b l y under the influence of different G S T e n z y m e s .  The  c o r r e s p o n d i n g G S H conjugates, n a m e l y 5 - G S - 4 - h y d r o x y V P A lactone a n d 5 - G S - 3 - e n e V P A , h a v e b e e n identified ( K a s s a h u n et a l . , 1 9 9 1 ; 1994).  In the present study, the  c y s t e i n y l g l y c i n e , glycine a n d N A C conjugates of 4 , 5 - e p o x y V P A a n d (E)-2,4-diene V P A w e r e c h a r a c t e r i z e d in addition to the G S H conjugates following a d o s e of 4 - e n e V P A to rats.  T h i s o b s e r v a t i o n is novel for 4 - e n e V P A m e t a b o l i s m but is in a c c o r d a n c e with  results d e r i v e d from a n earlier investigation c o n d u c t e d in o u r laboratory regarding Nformyl a m p h e t a m i n e metabolism to a G S H conjugate, w h e r e all metabolites a l o n g the mercapturic a c i d pathway w e r e a l s o detected in rat bile (Borel a n d Abbott, 1995). T h e liver is generally b e l i e v e d to b e the major site for G S H conjugation while the kidney a n d the s m a l l intestine a r e r e s p o n s i b l e for d e g r a d a t i o n of G S H c o n j u g a t e s to c y s t e i n e c o n j u g a t e s (Meister a n d T a t e , 1976; T a t e , 1980). C y s t e i n e c o n j u g a t e s formed in the kidney or gut a r e s u g g e s t e d to b e transported b a c k to the liver for A/-acetylation (Inoue et a l . , 1 9 8 2 ; 1984).  B e c a u s e the kidney w a s d e t e r m i n e d to h a v e a b u n d a n t N-  204  a c e t y l t r a n s f e r a s e activity, with a specific activity nearly twice that of liver, it is p u z z l i n g that  the  interorgan  model  for  mercapturic  acid  biosynthesis  requires  cysteine  c o n j u g a t e s to be A/-acetylated in the liver (Inoue et a l . , 1982). R e c e n t l y , the m o d e l w a s further c h a l l e n g e d by the finding that the G S H , cysteinylglycine, c y s t e i n e a n d N A C c o n j u g a t e s w e r e d e t e c t e d a s the biliary metabolites of C D N B in p e r f u s e d rat a n d g u i n e a pig livers ( H i n c h m a n et a l . , 1991). T h u s , a s a n alternative it h a s b e e n p r o p o s e d that the G S H conjugates c a n be d e g r a d e d within the biliary s p a c e to the c y s t e i n e c o n j u g a t e s , the latter being transported b a c k to the liver for A/-acetylation a n d the resultant mercapturic a c i d s r e l e a s e d into the bile ( H i n c h m a n a n d Ballatori, 1994).  The  on-line L C / M S / M S identification of all the mercapturic a c i d pathway c o n j u g a t e s derived from the m e t a b o l i s m of 4 - e n e V P A following a s i n g l e d o s e after bile duct c a n n u l a t i o n a p p e a r s to be in a c c o r d a n c e with this biliary-hepatic c y c l i n g m o d e l . F o r the s a k e of d i s c u s s i o n , the conjugated 4 - e n e V P A metabolites will b e divided into two g r o u p s , the 5-thiol-4-hydroxy V P A lactone s e r i e s a s group 1 a n d the 5-thiol-3e n e V P A s e r i e s a s group 2.  F r o m a quantitative  point of view, the intact G S H  c o n j u g a t e s w e r e still the major metabolites, making up 4 6 ~ 4 7 % in e a c h g r o u p of the biliary mercapturic a c i d pathway metabolites, w h e r e a s the c y s t e i n y l g l y c i n e conjugates r a n k e d s e c o n d , b e i n g 4 0 % in group 1 a n d 4 5 % in group 2. T h e s e large a m o u n t s of 5c y s g l y - 4 - h y d r o x y V P A lactone a n d 5 - c y s g l y - 3 - e n e V P A found in the bile c o n c e p t u a l l y contradict the a s s u m p t i o n that primary metabolites of G S H c o n j u g a t e s a r e f o r m e d a n d d e g r a d e d in the kidney or s m a l l intestine.  S i n c e most of the 7 - G T activity in rat liver  w a s f o u n d to be l o c a l i z e d in bile ductules ( H a h n et a l . , 1 9 7 8 ; Inoue et a l . , 1983),  205  hydrolysis of the G S H conjugates to the c o r r e s p o n d i n g c y s t e i n y l g l y c i n e c o n j u g a t e s may o c c u r at t h e s e distal sites within the biliary tree. T h e c y s t e i n e a n d N A C conjugates w e r e determined to be ~ 1 0 % in e a c h group of metabolites with the l e v e l s of the intermediate c y s t e i n e c o n j u g a t e s b e i n g h i g h e r than that  of  the  mercapturic  acetyltransferase.  acids,  suggesting  a  p o s s i b l e saturation  of  hepatic  N-  O n the other h a n d , b e c a u s e the e x p e r i m e n t s w e r e c o n d u c t e d in  intact a n i m a l s , the contribution from potential hepto-renal c y c l i n g to the profile of the d e t e c t e d biliary metabolites c o u l d not be e x c l u d e d .  T h e l e v e l s of N A C conjugates  might a l s o be d e p e n d e n t on the efficiency of the export s y s t e m that eliminates the c o n j u g a t e s from the liver.  N e v e r t h e l e s s , our o b s e r v a t i o n s regarding thiol conjugate  m e t a b o l i s m of two distinctive c l a s s e s of c o m p o u n d s , AMormyl a m p h e t a m i n e (Borel a n d Abbott,  1995) a n d 4 - e n e V P A , in rats m a y be v i e w e d a s a n indication that the  d e g r a d a t i o n of G S H c o n j u g a t e s to mercapturic a c i d s d o e s o c c u r in the liver a n d m a y be a rather c o m m o n p h e n o m e n o n for xenobiotics. T h i s c o n c l u s i o n is further s u p p o r t e d by a recent report in w h i c h detection of the mercapturic a c i d pathway metabolites of verlukast in rat bile w a s d e s c r i b e d (Nicoll-Griffith e t a l . , 1995). S i n c e biliary excretion of all mercapturic a c i d pathway metabolites is c o n s i d e r e d to be u n u s u a l , the a s s a y m e t h o d s u s e d in this study are noteworthy of d i s c u s s i o n in order to solidify our c l a i m . T h e L C / M S / M S m e t h o d o l o g y w a s in g e n e r a l c h a r a c t e r i z e d by its specificity towards the a n a l y t e s of interest.  H o w e v e r , a q u e s t i o n a r o s e w h e n it  w a s noticed that fragment ions might be p r o d u c e d prior to C I D in the Q 2 c e l l a n d thus a primary d a u g h t e r ion c o u l d be subjected to C I D to form s e c o n d a r y fragments.  206  For  e x a m p l e , the protonated m o l e c u l a r ions of the G S H c o n j u g a t e s (MH+: m/z 4 4 8 for both 5 - G S - 4 - h y d r o x y V P A lactone a n d 5 - G S - 3 - e n e V P A ) d i s s o c i a t e d through the l o s s of pyroglutamic  a c i d to give fragment  ions at m/z  319.  Such M S  neutral induced  d i s s o c i a t i o n might be c o n f u s e d with the ions arising from the metabolites of the G S H c o n j u g a t e s if the metabolite a n d the parent G S H conjugate itself h a p p e n e d to co-elute. T h i s p r o b l e m a r i s e s b e c a u s e the fragment  ions of the G S H c o n j u g a t e s a n d  the  protonated parent ions of their metabolites p o s s e s s identical c h e m i c a l structures a n d therefore w o u l d give identical daughter ion s p e c t r a upon C I D .  In this c a s e , fragment  ions at m/z 3 1 9 are virtually the s a m e a s the protonated c y s t e i n y l g l y c i n e conjugates d e r i v e d from the c o r r e s p o n d i n g G S H conjugates v i a the 7 - G T c a t a l y z e d r e m o v a l of the glutamyl r e s i d u e .  A similar situation may exist for the c y s t e i n e / N A C conjugate pairs:  the l o s s of k e t e n e from the N A C conjugates (MH+ = m/z 3 0 4 for both 5 - N A C - 4 - h y d r o x y V P A lactone a n d 5 - N A C - 3 - e n e V P A ) to give the fragment  at m/z 2 6 2 is a m a s s  equivalent to the protonated m o l e c u l a r ions of the c y s t e i n e c o n j u g a t e s .  If the cysteine  c o n j u g a t e s a n d their A/-acetylated metabolites w e r e to co-elute, s p e c i f i c detection of the cysteine  conjugates  fragmentation  would  be  difficult  at  best.  cysteine  a  decrease  in  the  prior to C I D in the Q 2 cell c o u l d be a c h i e v e d by r e d u c i n g the c o n e  v o l t a g e , the sensitivity would be c o m p r o m i s e d . the  Although  conjugates  from  the  NAC  T h u s , c h r o m a t o g r a p h i c s e p a r a t i o n of conjugates  and  separation  of  the  c y s t e i n y l g l y c i n e c o n j u g a t e s from the G S H conjugates w e r e sought in o r d e r to u n ambiguously  identify  these  metabolites.  Under  current  conditions,  HPLC  c h r o m a t o g r a p h y w a s d e v e l o p e d s u c h that no o v e r l a p p i n g of p e a k s o c c u r r e d a m o n g  207  t h o s e c o m p o u n d s w h i c h m a y potentially p r o d u c e c o m m o n fragments of interest (Figure 3.12 a n d 3.13). S i m i l a r to that found for (E)-2,4-diene V P A treated rats ( S e c t i o n s 3.3.1 a n d 4.3), two structural i s o m e r s of the G S H conjugates of (E)-2,4-diene V P A w e r e p r o d u c e d during the m e t a b o l i s m of 4 - e n e V P A in rats. A l o n g with 5 - G S - 3 - e n e V P A , 5 - G S - 2 - e n e V P A w a s identified a s a minor biliary metabolite. T h i s result w a s c o n s i s t e n t with our in vitro investigation u s i n g the A/-acetylcysteamine thioester of ( E ) - 2 , 4 - d i e n e V P A a s a structural mimic of the c o r r e s p o n d i n g C o A ester. A s m a l l amount of 5 - G S - 2 - e n e V P A A/-acetylcysteamine ester w a s detected w h e n rat liver mitoplast fraction w a s u s e d a s the s o u r c e of G S T e n z y m e (Section 3.4.2).  C o n j u g a t i o n of G S H to ( E ) - 2 , 4 - d i e n e V P A  d e r i v e d from 4 - e n e V P A is b e l i e v e d to o c c u r in mitochondria with the d i e n e metabolite activated for G S H addition by its C o A thioester form ( S c h e m e 4.3) ( K a s s a h u n et a l . , 1991). O t h e r biliary G S H related metabolites found c o m m o n for both 4 - e n e V P A a n d (E)-2,4-diene  VPA  were  5-GS-3-ene  VPA-glucuronide and  5-NAC-3-ene VPA-  g l u c u r o n i d e . In contrast to (E)-2,4-diene V P A metabolism w h e r e the G S H - g l u c u r o n i d e di-conjugate in bile w a s 6-fold that of the c o r r e s p o n d i n g G S H m o n o - c o n j u g a t e (Section 3.3.4), the l e v e l s of 5 - G S - 3 - e n e V P A w e r e determined to be 10-times h i g h e r than that of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e in 4 - e n e V P A treated rats.  In order to e x p l a i n this  o b s e r v a t i o n , o n e must c o n s i d e r that the conjugation of G S H with the d i e n e substrate o c c u r s primarily in e n d o p l a s m i c reticulum upon a d o s e of (E)-2,4-diene V P A . W h e t h e r the m o n o - G S H conjugate found in the bile of rats administered ( E ) - 2 , 4 - d i e n e V P A w a s / /  208  a product of the p - g l u c u r o n i d a s e c a t a l y z e d hydrolysis of 5 - G S - 3 - e n e V P A - g l u c u r o n i d e or resulted from a C o A mediated p r o c e s s in mitochondria r e m a i n s to b e clarified. But, this quantitative c o m p a r i s o n of the di-conjugate v s . the mono-conjugate arising from 4e n e V P A or from (E)-2,4-diene V P A supports the postulation that the G S H - g l u c u r o n i d e di-conjugate found in 4 - e n e V P A metabolism is likely formed v i a the glucuronidation of (E)-2,4-diene V P A " l e a k e d " out from the mitochondria followed by conjugation with G S H in the c y t o s o l i c compartment ( S c h e m e 4.3).  T h e 5 - G S - 3 - e n e V P A - g l u c u r o n i d e is  s u b s e q u e n t l y c o n v e r t e d to the c o r r e s p o n d i n g N A C - g l u c u r o n i d e di-conjugate  through  the biliary-hepatic cycling pathway for mercapturic a c i d b i o s y n t h e s i s . In the urine of rats treated with 4 - e n e V P A , only the N A C c o n j u g a t e s of 4 , 5 e p o x y V P A a n d (E)-2,4-diene V P A a n d the N A C - g l u c u r o n i d e di-conjugate of (E)-2,4diene V P A were detected.  T h i s result c o u l d be attributed to the s e l e c t i v e transport  m e c h a n i s m s of the kidney (Dekant et a l . , 1988). Quantitatively, total biliary excretion of the G S H related conjugates, including the di-conjugate, o v e r a 6 h period d e t e r m i n e d to be 5 % of the 4 - e n e V P A d o s e .  was  T h i s result is at the lower limit of the  reported 5 - 1 2 % range estimated from intensities of M S / M S  r e s p o n s e s for  c o n j u g a t e s after 4 - e n e V P A administration ( K a s s a h u n et a l . , 1994).  thiol  O u r d a t a further  confirm that bioactivation of 4 - e n e V P A o c c u r s in the liver to form highly  reactive  electrophilic metabolites w h i c h react with hepatic G S H a n d h a v e potential to attack critical m a c r o m o l e c u l e s ( K a s s a h u n et a l . , 1994; S e c t i o n 3.3.8).  209  210  4.2. Evaluating the Ability of (E)-2,4-Diene VPA to Alkylate Peptides and Proteins  In addition to s e q u e s t r a t i o n of cellular G S H , the C o A thioester of (E)-2,4-diene VPA  m a y directly attack critical b i o m a c r o m o l e c u l e s to form c o v a l e n t bond(s)  thereby  p r o d u c e toxicity  ( K a s s a h u n et a l . , 1994).  T h i s capability of the  and diene  metabolite to alkylate peptides or proteins w a s clearly illustrated in this study by u s i n g r e d u c e d oxytocin a s a m o d e l biological substrate.  T h e 2 , 4 - d i e n e V P A - N A C A reacted  stoichiometrically with the c y s t e i n e r e s i d u e s o n r e d u c e d oxytocin, s u g g e s t i n g that free thiols o n other p o l y p e p t i d e s or proteins will be v u l n e r a b l e to attack.  This observation  a p p e a r e d consistent with previous findings w h e r e b y c o v a l e n t protein binding to the isolated mitochondrial fraction w a s parallel to the formation of (E)-2,4-diene V P A a n d w a s C o A d e p e n d e n t ( K a s s a h u n et a l . , 1994). Similarly, the reactive 2,4-diene V P A - g l u c u r o n i d e c o u l d attack proteins a s well. Irreversible binding to rat liver proteins w a s reported to o c c u r with both r a d i o l a b e l e d V P A a n d 4 - e n e V P A in vivo a n d in vitro, a n d the incorporation of radiolabel into proteins  was  o b s e r v e d to  be  d e c r e a s e d in  rat  hepatocytes when  glucuronidation w e r e a d d e d into the incubation ( P o r u b e k et a l . , 1989).  inhibitors  of  In the present  study, 2 , 4 - d i e n e V P A - g l u c u r o n i d e w a s demonstrated to react with r e d u c e d oxytocin, resulting  in  an  adduct  in  which  the  bound  stoichiometric to the a v a i l a b l e c y s t e i n e r e s i d u e s . t h e s e o b s e r v a t i o n s to V P A hepatotoxicity  211  2,4-diene  VPA-glucuronide  was  T h e toxicological s i g n i f i c a n c e of  r e m a i n s to be e s t a b l i s h e d , a n d further  investigations m a y n e e d to f o c u s o n the identification a n d c h a r a c t e r i z a t i o n of proteins that are targeted by (E)-2,4-diene V P A .  4.3. Conjugation of GSH with (E)-2,4-Diene VPA Catalyzed by Rat Liver GST Enzymes  D e s p i t e a n u m b e r of G S H c o n j u g a t e s being detected during V P A m e t a b o l i s m , no direct e v i d e n c e h a s b e e n reported to s u g g e s t the involvement of G S T e n z y m e s .  This  study w a s d e s i g n e d to investigate the role of G S T in c a t a l y z i n g the M i c h a e l addition of G S H to (E)-2,4-diene V P A , a toxic V P A metabolite. W h e n the isolated rat liver c y t o s o l or m i t o c h o n d r i a w e r e i n c l u d e d in the incubations containing G S H a n d 2 , 4 - d i e n e V P A N A C A , both s u b c e l l u l a r fractions i n c r e a s e d the production of G S H c o n j u g a t e s .  The  G S T activities in t h e s e isolated fractions w e r e clearly d e m o n s t r a t e d in a positive control experiment u s i n g C D N B a s a "general substrate" for t h e s e e n z y m e s ( M a n n e r v i k a n d D a n i e l s o n , 1988). T h e o b s e r v e d G S T activities are thus b e l i e v e d to b e r e s p o n s i b l e for the i n c r e a s e s obtained in the reaction rates of G S H with 2 , 4 - d i e n e  VPA-NACA.  A d d i t i o n a l e v i d e n c e for this claim i n c l u d e s , 1) the catalytic effects w e r e a b o l i s h e d w h e n the s u b c e l l u l a r fractions w e r e boiled a n d 2) parallel results w e r e o b t a i n e d with partially purified G S T e n z y m e . T h e s e c o n d a c h i e v e m e n t of this investigation is in regard to s p e c u l a t i o n that formation of the C o A thioester of (E)-2,4-diene V P A w a s a pre-requisite for conjugation with G S H . T h i s w a s a c c o m p l i s h e d by u s i n g a structural mimic of the C o A thioester,  212  namely 2,4-diene V P A - N A C A .  T h e r e w a s no detectable  reaction in  incubations  involving G S T , G S H a n d (E)-2,4-diene V P A a s the free a c i d , w h e r e a s the conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A p r o c e e d e d at a m e a s u r a b l e rate e v e n with a 10-fold l e s s amount of the e n z y m e . T h u s , the esterification of (E)-2,4-diene V P A is e s s e n t i a l for its conjugation with G S H e v e n in the p r e s e n c e of G S T . In other w o r d s , only the G S H w a s activated by G S T , w h i c h is in a g r e e m e n t with the p r o p o s e d m e c h a n i s m of action for this e n z y m e . E s t e r forms of the d i e n e other than the C o A thioester m a y a l s o contribute to the conjugation with G S H (Section 3.3.2). T h e s o u r c e of the G S T activity detected in our mitoplast p r e p a r a t i o n s n e e d s to be a d d r e s s e d , simply b e c a u s e G S T activity in cytosol is 10-fold higher than that s e e n in m i t o c h o n d r i a ( A d d y a et a l . , 1994).  B a s e d on the s p e c i f i c activities of the cytosolic  m a r k e r e n z y m e , L D H , the s u s p e n s i o n of intact mitochondria w a s e s t i m a t e d to contain 2%  of  cytosolic  contamination  contaminant.  An  additional  5-fold  decrease  level w a s a c h i e v e d following a digitonin treatment  in  the  apparent  a n d three  more  w a s h e s to p r o d u c e a negligible interference from cytosol to the m e a s u r e d mitochondrial G S T activity. differences  M o r e c o n v i n c i n g e v i d e n c e w a s provided by the o b s e r v e d 5 - 1 5 fold  in G S T activities  between the  supernatants  disrupted mitoplasts a n d t h o s e from the intact mitoplasts.  isolated from  the  sonic  T h u s , the d e t e c t e d G S T  activity a p p e a r e d to be a s s o c i a t e d largely within the mitochondrial matrix ( A d d y a et a l . , 1994). H o w e v e r , other possibilities c a n not be eliminated, b e c a u s e it w a s reported that G S T r e l e a s e did not parallel that of the mitochondrial matrix marker e n z y m e s following digitonin treatment (Ryle a n d M a n t l e , 1984).  213  T h e G S T c a t a l y z e d M i c h a e l addition of G S H to 2 , 4 - d i e n e V P A - N A C A resulted in two structural i s o m e r s , n a m e l y 5 - G S - 3 - e n e V P A - N A C A v i a 1,6-addition a n d 5 - G S - 2 e n e V P A - N A C A from 5,6-addition of G S H to the d i e n e .  It is likely that G S " thiolate  a n i o n a r i s i n g from the G S T promoted deprotonation of G S H (Armstrong, 1991) attacks the terminal d o u b l e b o n d of 2 , 4 - d i e n e V P A - N A C A to g e n e r a t e intermediates with a n e g a t i v e c h a r g e s t a b i l i z e d through r e s o n a n c e a n d l o c a l i z e d on either the adjacent c a r b o n c e n t e r or o n the remote c a r b o n y l o x y g e n atom.  C o m p l e t i o n of the reaction is  a c c o m p l i s h e d by capture of a proton by the a n i o n i c intermediate from either the G S T e n z y m e , or simply from the a q u e o u s m e d i u m ( S c h e m e 4.4). A n interaction b e t w e e n a G l u or A s p r e s i d u e of the e n z y m e a n d the c a r b o n y l g r o u p of 2 , 4 - d i e n e V P A - N A C A w o u l d i n c r e a s e the nucleophility of the d i e n e a n d therefore facilitate the conjugation with G S H ( S c h e m e 4.4) (Mannervik a n d D a n i e l s o n , 1988). In a similar f a s h i o n , a water m o l e c u l e m a y s e r v e to promote the s p o n t a n e o u s reaction o c c u r r i n g at p H 6.7 b e t w e e n G S H a n d the d i e n e , the difference from the G S T c a t a l y z e d reaction b e i n g that the 1,6addition product w a s found to be highly predominant. B e c a u s e the intermediate h a v i n g a n e g a t i v e c h a r g e on the c a r b o n atom is c o n s i d e r e d to be l e s s f a v o r e d than w h e n on o x y g e n ( S c h e m e 4.4), this o b s e r v e d distinction in product formation b e t w e e n the e n z y m e c a t a l y z e d a n d the s p o n t a n e o u s reaction c a n be interpreted in terms of the e n z y m e acting a s a n effective proton donor to capture the short-lived intermediate a n d thus a c c o m p l i s h the formation of the 5,6-addition product. The higher  in  product ratio of 5 - G S - 2 - e n e V P A - N A C A to 5 - G S - 3 - e n e V P A - N A C A w a s reactions  involving  cytosol than  214  in  those  involving  mitoplasts.  PB  pretreatment t e n d e d to favor i n c r e a s e d formation of the 5,6-addition i s o m e r (Table 4). O n e c o u l d s p e c u l a t e that different c l a s s e s of G S T e n z y m e s are r e s p o n s i b l e for the o b s e r v e d differences in product ratio. W h e t h e r the s m a l l amount of 5 - G S - 2 - e n e V P A N A C A d e t e c t e d in the mitoplast promoted reaction a r i s e s from c y t o s o l i c contamination or is the g e n u i n e product of mitochondrial G S T c a t a l y s i s requires further investigation. C o n s i s t e n t with the in vitro results, two structural i s o m e r s of the G S H conjugate of ( E ) - 2 , 4 - d i e n e V P A w e r e identified in the bile of rats a d m i n i s t e r e d ( E ) - 2 , 4 - d i e n e V P A . In this respect, the in vivo d a t a a p p e a r e d to support our in vitro s y s t e m , particularly the mitoplast promoted reaction. B a s e d on the intensity of M S / M S r e s p o n s e s , the biliary 5G S - 2 - e n e V P A concentration w a s estimated to be l e s s than that of its structural isomer. O n e c o u l d rationalize that a portion of the d o s e d (E)-2,4-diene V P A is t a k e n up by m i t o c h o n d r i a , within w h i c h the C o A thioester of the d i e n e reacts with G S H to form 5G S - 2 - e n e V P A a n d 5 - G S - 3 - e n e V P A v i a a G S T mediated p r o c e s s with the 1,6 addition b e i n g predominant ( K a s s a h u n et a l . , 1991). A l t h o u g h the characterization of 5 - G S - 3 - e n e V P A in rats w a s reported previously ( K a s s a h u n et a l . , 1 9 9 1 ; 1994), this is the first identification of 5 - G S - 2 - e n e V P A .  In  patients o n V P A therapy, a n unidentified N A C conjugate of ( E ) - 2 , 4 - d i e n e V P A w a s d e t e c t e d in addition to 5 - N A C - 3 - e n e V P A ( K a s s a h u n et a l . , 1991). T h u s , the d i s c o v e r y of 5 - G S - 2 - e n e V P A conjugate here provides a logical e x p l a n a t i o n for the  mystery  G C / M S p e a k o b s e r v e d in the patient urine extracts, i.e. 5 - N A C - 2 - e n e V P A . W h e t h e r the a p p a r e n t e q u a l intensity of the two isomeric N A C c o n j u g a t e s found in patient urine ( K a s s a h u n et a l . , 1991) represents a g e n u i n e result d e r i v e d from the m e t a b o l i s m of  215  V P A or a n artifact p r o d u c e d during the w o r k u p of urine s a m p l e s for G C / M S a s s a y is still not k n o w n . GST  e n z y m e s are known to be inducible by d r u g s , o n e of w h i c h is the  a n t i c o n v u l s a n t agent P B that is s o m e t i m e s u s e d with V P A in therapy ( A n i y a et a l . , 1993;  Foliot a n d  Beaune,  1994).  The  present  investigation  d e m o n s t r a t e d that  treatment of rats with P B p r o d u c e d i n c r e a s e d G S T activities in both the c y t o s o l i c a n d mitoplast fractions w h e n activities were estimated u s i n g C D N B a s s u b s t r a t e . T h e rate of conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A w a s a l s o e n h a n c e d in the hepatic c y t o s o l i c fraction isolated from P B treated a n i m a l s . P B is a l s o a well k n o w n i n d u c e r of m i c r o s o m a l P 4 5 0 e n z y m e s ( O k e y , 1990), a n d this effect h a s b e e n implicated in the high i n c i d e n c e of V P A - a s s o c i a t e d hepatotoxicity o b s e r v e d in the patients w h o were u n d e r polytherapy s i n c e a n e l e v a t e d P 4 5 0 activity c o u l d result in greater a m o u n t s of toxic metabolites during the biotransformation of V P A ( L e v y et a l . , 1990).  Because  G S H conjugation c a n be o n e of either detoxification or toxification, d e p e n d i n g on the nature a n d a m o u n t of the toxic s u b s t a n c e a s well a s cellular G S H status, the role of the P B inducible c y t o s o l i c G S T e n z y m e s in V P A hepatotoxicity r e m a i n s to be clarified.  216  <  CD JD  >  O  L  < O <  < O <  0  < >  < >  < >  z  < < z  z  CD  CD  c  CD  c  CD  C  CD  I  CD CO  I  CO  OJ CO  I  co co CD CD  CO  CD  CD  l  LO  1 LO  I  LO  X  .i S "O >-  *s  CM O JZ © •4-*  I  CO CD M—  O  c  N  £_  § CL 0 5  0  JZ  -4—'  ^  .2 CD  c UJ I  c o o t3  CD N  X  N C  CO  0  C  CD >  o  "E  CO  £  co CO CD CL >, C / J o "CO  UJ  I  •+-• o - CD I- < o CO CD  CD  O  CM  X  II  < DC  0 0 c 5= CO 0 ZJ JO E CO CO CO CO 3 ' c CD o 0 CO JZ LO  o •a CD c  E  H  o o CO  c UJ -  DQ  0 •a 0 CO o CL o  cr CO  CO  <<  o o << << CL CL > 0 c 0 CVJ  (3  ZJ  > 0 c 0  t3 1  CO CD cvi" 0  LO t 3  E 0 0  > CO  c  CO  X CO o CO o CD JZ  217  In contrast to the cytosolic fraction, the effect of P B on the mitoplast promoted conjugation of G S H with 2 , 4 - d i e n e V P A - N A C A w a s not apparent.  This observed  distinction b e t w e e n cytosol a n d mitochondria m a y relate to different G S T c l a s s e s , b e c a u s e G S T i s o e n z y m e s are known to h a v e different substrate p r e f e r e n c e s a n d to be i n d u c e d to different l e v e l s by P B (Pickett a n d L u , 1989).  O n the other h a n d , it is  p o s s i b l e that a n e n h a n c e d G S T catalytic effect in mitochondria w a s m a s k e d by a relatively higher protein concentration in the mitoplast fraction.  In a c c o r d with this  h y p o t h e s i s , purified mitochondrial G S T e n z y m e s w e r e s h o w n to h a v e s p e c i f i c activities c o m p a r a b l e to their cytosolic counterparts ( A d d y a et a l . , 1994), but the determined  in c r u d e  mitoplasts w e r e  only  1/10  that of c r u d e c y t o s o l .  activities Further  investigations m a y n e e d to f o c u s o n the influence of P B on purified mitochondrial G S T enzymes. An  impairment  of the  G S H redox s y s t e m within m i t o c h o n d r i a  d u e to  c h a l l e n g e of toxic c o m p o u n d s is known to result in cellular d a m a g e ( R e e d , 1990).  the A  r e d u c e d level of mitochondrial G S H h a s b e e n p r o p o s e d a s o n e p o s s i b l e m e c h a n i s m l e a d i n g to V P A - a s s o c i a t e d hepatotoxicity ( K a s s a h u n et a l . , 1 9 9 1 ; 1994).  In vivo, the  administration of 4 - e n e V P A to rats d e c r e a s e d hepatic mitochondrial G S H to 7 0 % of control ( K a s s a h u n et a l . , 1994; S e c t i o n 3.6) a n d p r o d u c e d s e v e r e liver m i c r o v e s i c u l a r s t e a t o s i s ( K e s t e r s o n et a l . , 1984; S e c t i o n 3.6).  Bioactivation of 4 - e n e V P A in  m i t o c h o n d r i a to (E)-2,4-diene V P A C o A thioester w h i c h c a n react with G S H locally m a y a c c o u n t for the o b s e r v e d depletion of G S H a n d the a s s o c i a t e d toxicity ( K a s s a h u n et a l . , 1994; S e c t i o n 3.6). In this study, it w a s d e m o n s t r a t e d that conjugation of G S H with 2,4-  218  d i e n e V P A - N A C A w a s c a t a l y z e d by mitochondrial G S T , s u g g e s t i n g that this e n z y m e ( s ) p r o m o t e s depletion of mitochondrial G S H by (E)-2,4-diene V P A .  4.4. Preliminary Metabolic Studies of a-Fluorinated VPA Analogues in Rats  Not only d o e s mitochondrial p-oxidation represent a major p h a s e I pathway during V P A biotransformation, but this pathway m a y a l s o contribute significantly to metabolic  activation  of the  drug  ( K a s s a h u n et  a l . , 1 9 9 1 ; 1994; S e c t i o n  1.2.2).  Fluorination of V P A a n d 4 - e n e V P A at the a - p o s i t i o n s w a s d e s i g n e d to a v o i d the participation of p-oxidation in the metabolism of the resulting a n a l o g u e s . A s u b s e q u e n t investigation of the metabolism of a-fluoro V P A a n d a - f l u o r o - 4 - e n e V P A in the rat m o d e l w a s c o n d u c t e d in order to validate our initial h y p o t h e s i s . In a preliminary in vivo study, it w a s evident in rats that the e x p e c t e d interruption of drug m e t a b o l i s m v i a p-oxidation w a s a c h i e v e d by a-fluorination of V P A a n d 4 - e n e VPA.  T h e primary p-oxidation metabolites, n a m e l y ( E ) - 2 - e n e V P A a n d (E)-2,4-diene  V P A , w e r e not detected in the s e r u m a n d urine of rats treated with a-fluoro V P A a n d a fluoro-4-ene V P A , respectively. T h i s observation a p p e a r e d consistent with the c o n c e p t that a strong C - F b o n d resists m a n y e n z y m a t i c reactions ( W e l c h , 1990).  But, s e v e r a l  factors w h i c h are not related to the C - F b o n d strength c o u l d be involved in the apparent interruption of p-oxidative m e t a b o l i s m .  F o r e x a m p l e , the drug m a y b e i n c a p a b l e of  forming a C o A thioester; or m a y not be taken up by mitochondria.  219  T h e m e t a b o l i s m of a-fluoro V P A in the rat w a s o b s e r v e d to give a-fluoro-4-ene VPA.  T h i s biotransformation, b a s e d on the k n o w e d g e of V P A m e t a b o l i s m to 4 - e n e  V P A (Rettie et a l . , 1987; 1988), is thought to be mediated by m i c r o s o m a l P 4 5 0 .  The  P 4 5 0 abstracts a hydrogen from the fluorinated V P A a n a l o g u e to g e n e r a t e a free radical intermediate which partitions between recombination with o x y g e n s p e c i e s a n d elimination of a s e c o n d proton with the latter p r o c e s s p r o d u c i n g the terminal olefin. similar m e c h a n i s m c o u l d be a p p l i e d to the formation m e t a b o l i s m ( S c h e m e 1.4).  A  of 3 - e n e V P A from V P A  H o w e v e r , neither a-fluoro-3-ene V P A nor its s p o n t a n e o u s  b r e a k d o w n product 4 - h y d r o x y - 2 - e n e V P A lactone w a s detected in the urine of rat treated with a-fluoro V P A . T h e lack of a-fluoro-3-ene V P A during the m e t a b o l i s m of a fluoro V P A m a y s u g g e s t that m i c r o s o m a l P 4 5 0 is not involved in the m e t a b o l i s m of V P A to 3 - e n e V P A , although the p r e s e n c e of the fluorine substituent  c o u l d alter  the  biotransformation p a t h w a y s ( M a c d o n a l d , 1982) T h e major p h a s e II metabolites in rats treated with a-fluoro V P A or a-fluoro-4e n e V P A w e r e the c o r r e s p o n d i n g glutamine conjugates, b a s e d o n the intensity of G C / M S r e s p o n s e s o b s e r v e d for the urine extracts. A m i n o a c i d conjugation is g e n e r a l l y r e g a r d e d a s a C o A mediated p r o c e s s occurring in mitochondria (Hutt a n d C a l d w e l l , 1990).  In this respect, a-fluorinated V P A a n a l o g u e s are very likely to b e t a k e n up by  mitochondria, forming the c o r r e s p o n d i n g C o A thioesters which preferentially react with L-glutamine. W h e t h e r the C o A thioesters of the fluorinated a n a l o g u e s interact with the P-oxidative e n z y m e s r e m a i n s to be clarified.  220  B a s e d o n t h e s e d a t a , it is apparent that a-fluorination b l o c k s p-oxidation of the resulting V P A a n a l o g u e s a s e x p e c t e d . Further investigation of the a-fluorinated V P A a n a l o g u e s with regards to m e t a b o l i s m , toxicology a n d anticonvulsant activities  in  c o m p a r i s o n to their non-fluorinated counterparts a p p e a r e d to be worthy of pursuit.  4.5. Comparative Toxicological Studies of 4-Ene V P A and a-Fluoro-4-ene V P A in Rats  T h e hepatotoxicity of V P A is thought to be a s s o c i a t e d with a metabolic p r o c e s s that b e g i n s with the formation of the m i c r o s o m a l P 4 5 0 metabolite 4 - e n e V P A w h i c h is a structural a n a l o g u e of the known hepatotoxicant, 4 - P A ( G e r b e r et a l . , 1979).  The  p r o p o s e d m e c h a n i s m for V P A toxicity s u g g e s t s that the production of hepatotoxicity is highly d e p e n d e n t o n the formation  of reactive intermediates from 4 - e n e V P A in  mitochondria, namely, (E)-2,4-diene V P A a n d 3-keto-4-ene V P A , the first of w h i c h is a potential G S H depleting agent a n d the s e c o n d is a putative inhibitor of  p-oxidation  e n z y m e s (Baillie, 1988; K a s s a h u n et a l . , 1991) ( S c h e m e 1.6, B). T h i s m e c h a n i s m for V P A hepatotoxicity is b a s e d largely on prior k n o w l e d g e that 4 - P A , w h e n m e t a b o l i z e d by P-oxidation v i a (E)-2,4-pentadienoic a c i d , p r o d u c e s 3 - o x o - 4 - p e n t e n o i c a c i d that inhibits the e n z y m e ( s ) of that pathway in a suicidal m a n n e r ( S c h u l z , 1983). H o w e v e r , there are f u n d a m e n t a l differences in the metabolism of 4 - e n e V P A c o m p a r e d to 4 - P A in rats, the m e c h a n i s m s that c a u s e liver injury may not be identical for t h e s e two hepatotoxicants ( K a s s a h u n a n d Abbott, 1993). F o r e x a m p l e , (E)-2,4-diene V P A a n d its N A C conjugate  221  a r e predominant metabolites of 4 - e n e V P A in the rat while 3 - k e t o - 4 - e n e V P A w a s difficult to detect.  T h e reverse w a s true for c o r r e s p o n d i n g metabolites of 4 - P A  ( K a s s a h u n a n d Abbott, 1993). But, apart from the m e t a b o l i s m details, it is r e a s o n a b l e to  p r o p o s e that bioactivation through  p-oxidation  is a c o m m o n feature  for  both  c o m p o u n d s a n d that hepatotoxicity of either 4 - e n e V P A or 4 - P A c a n be p r e v e n t e d by averting their m e t a b o l i s m in mitochondria. In this study, it w a s c o n f i r m e d that the major metabolites of 4 - e n e V P A e x c r e t e d in rats w e r e (E)-2,4-diene V P A w h i c h is a key intermediate in the putative bio-activated toxification p r o c e s s ( S c h e m e 1.6, B) a n d the NAC  conjugate  of (E)-2,4-diene V P A which a r i s e s from  conjugate ( K a s s a h u n et a l . , 1991).  the  corresponding G S H  N o n e of t h e s e s p e c i e s c o u l d be d e t e c t e d in the  urine a n d s e r u m of rats treated with the a-fluorinated c o m p o u n d . A s a c o n s e q u e n c e of preventing the m e t a b o l i s m of a-fluoro-4-ene V P A by the p-oxidative pathway,  this  c o m p o u n d , a s m e a s u r e d by liver s t e a t o s i s , w a s determined to be n o n h e p a t o t o x i c in rats.  F o r e x a m p l e , in contrast to the more than 8 5 % of h e p a t o c y t e s affected in rats  treated with 4 - e n e V P A , hepatic m i c r o v e s i c u l a r s t e a t o s i s w a s a b s e n t in the a n i m a l s a d m i n i s t e r e d oc-fluoro-4-ene V P A . P a r a l l e l results w e r e s e e n w h e n 4 - P A a n d F 2 - 4 - P A w e r e c o m p a r e d . Substitution of the cc-hydrogens of 4 - P A with two fluorine a t o m s w a s similarly e x p e c t e d to prevent metabolism  of  F2-4-PA  through  the  p-oxidation  pathway.  While  the  block  of  m e t a b o l i s m w a s not c o n f i r m e d , F 2 - 4 - P A , like oc-fluoro-4-ene V P A , w a s ineffective in p r o d u c i n g m i c r o v e s i c u l a r s t e a t o s i s in rat liver, e v e n though 4 - P A w a s a potent i n d u c e r of  such  lesions.  T a k e n together,  these  222  results  strongly  s u g g e s t that  reactive  metabolites p r o d u c e d from (3-oxidative p r o c e s s e s in mitochondria are d e c i s i v e factors in 4 - P A - a n d 4 - e n e V P A - r e l a t e d liver toxicities. In order to further clarify a b a s i s for the apparent differences of fluorinated a n d nonfluorinated derivatives to p r o d u c e liver toxicity in rats, the effects of 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A on hepatic G S H levels w e r e e x a m i n e d .  T h e r e are two p o o l s of  cellular G S H , o n e located in cytosol a n d the other in mitochondria ( R e e d , 1990) a n d it is important for m e c h a n i s t i c c o n s i d e r a t i o n s of a toxic s u b s t a n c e to distinguish which s o u r c e s of G S H are being d e p l e t e d .  B e c a u s e mitochondria are b e l i e v e d to lack  c a t a | a s e , G S H c o u p l e d with G S H p e r o x i d a s e is c o n s i d e r e d to b e the only antioxidant d e f e n s e in this o r g a n e l l e ( M a r t e n s s o n a n d Meister, 1 9 8 9 ; R e e d , 1990).  Although  recent d a t a indicate that c a t a l a s e is present in rat heart mitochondria ( R a d i et a l . , 1991), this e n z y m e cannot act a s a d e f e n s e against p e r o x i d e s other than h y d r o g e n peroxide.  F o r e x a m p l e , it w a s reported that hepatic mitochondrial G S H w a s markedly  r e d u c e d in rat h e p a t o c y t e s by (R,S)-3-hydroxy-4-pentenoate ( S h a n et a l . , 1993). T h e c a u s a t i v e s p e c i e s in this p r o c e s s is b e l i e v e d to be the metabolite 3 - o x o - 4 - p e n t e n o a t e ( S h a n et a l . , 1993) which is a l s o a metabolite of 4 - P A ( K a s s a h u n a n d Abbott, 1993). A s a c o n s e q u e n c e of the G S H depletion by 3-hydroxy-4-pentenoate, the toxicity of tbutyl h y d r o p e r o x i d e to h e p a t o c y t e s w a s significantly potentiated ( S h a n et a l . , 1993). In the present study,  a-fluoro-4-ene V P A did in fact d e c r e a s e total hepatic G S H , but it  h a d no effect on G S H within mitochondria. T h e acute administration of 4 - e n e V P A , on the other h a n d , significantly r e d u c e d mitochondrial a s well a s total hepatic G S H l e v e l s .  223  A similar depletion effect of 4 - e n e V P A on liver G S H h a s recently b e e n reported by Baillie a n d c o - w o r k e r s ( K a s s a h u n et a l . , 1994). T h e o b s e r v e d distinction in mitochondrial G S H depletion b e t w e e n 4 - e n e V P A and  oc-fluoro-4-ene  V P A could  be  due  entirely  to  differences  in  metabolism.  C o n j u g a t i o n of the mitochondrial metabolites of 4 - e n e V P A , n a m e l y ( E ) - 2 , 4 - d i e n e V P A a n d to a m u c h l e s s e r extent 3-keto-4-ene V P A , with G S H residing within mitochondria m a y a c c o u n t for the apparent depletion of this critical G S H pool ( S e c t i o n 3.3.3). Similarly the lack of effect of oc-fluoro-4-ene V P A on mitochondrial G S H l e v e l s is in a c c o r d with the a b s e n c e of (E)-2,4-diene V P A a s a metabolite.  Therefore,  the  n o n s t e a t o g e n i c properties of oc-fluoro-4-ene V P A together with the a b s e n c e of effects on  mitochondrial  G S H support  the  hypothesis  that  the  selective  depletion  of  mitochondrial G S H p l a y s a critical role in the hepatotoxicity of 4 - e n e V P A , a n d by e x t e n s i o n V P A . It h a s b e e n reported, for e x a m p l e , that V P A w a s c a p a b l e of d e c r e a s i n g G S H l e v e l s in rat liver ( J e z e q u e l , A . M . et a l . , 1984) a n d in certain c e l l lines ( S i m o n et a l . , 1994).  T h e urinary N A C conjugate of (E)-2,4-diene V P A w a s found to b e higher  than a v e r a g e in patients w h o h a d d e v e l o p e d liver toxicity while o n V P A therapy ( K a s s a h u n et a l . , 1991) a n d in s o m e c a s e s patients with V P A a s s o c i a t e d hepatotoxicity w e r e s u c c e s s f u l l y treated with s u p p l e m e n t s of N A C (Farrell a n d Abbott, 1991).  The  latter w a s b e l i e v e d to i n c r e a s e intracellular G S H levels (Farrell a n d Abbott, 1991). T h e depletion of cytosolic G S H probably a c c o u n t s for most of the d e c r e a s e of total hepatic G S H o b s e r v e d for 4 - e n e V P A in this study (Table 7) a n d is largely the result of conjugation of cytosolic G S H to 2-(2'-carboxypentanyl)oxirane  224  (4,5-epoxy  V P A ) , a m i c r o s o m a l P 4 5 0 mediated oxidation product of 4 - e n e V P A m e t a b o l i s m ( K a s s a h u n et a l . , 1994; S e c t i o n 3.3.3) ( S c h e m e 1.6, B). fluoro-4-ene  V P A by  microsomal  P450  to  L i k e w i s e , m e t a b o l i s m of oc-  2-fluoro-4,5-epoxy  V P A followed  by  conjugation with G S H w o u l d a c c o u n t for the apparent d e c r e a s e of total hepatic G S H s e e n in rats treated with this fluorinated c o m p o u n d (Table 7). T h e e p o x i d e metabolite of 4 - e n e V P A m a y a l s o be important to the o b s e r v e d hepatotoxicity of 4 - e n e V P A although the results with the fluorinated derivative w o u l d s u g g e s t that the reaction of e p o x i d e with G S H is of l e s s s i g n i f i c a n c e to the production of s t e a t o s i s .  It is not fully  u n d e r s t o o d at this time why in the c a s e of 4 - e n e V P A treated rats the d e c r e a s e in mitochondrial G S H w a s not detected until at least 4 5 min p o s t - d o s e w h e r e a s total G S H levels w e r e a l r e a d y r e d u c e d to - 7 0 % of control (Table 7 a n d 8).  O n e might a s s u m e  that this is the time n e e d e d for 4 - e n e V P A to c r o s s mitochondrial m e m b r a n e s , to be m e t a b o l i z e d to (E)-2,4-diene V P A - C o A thioester, a n d to react with the r e s i d i n g G S H ( S e c t i o n 4.7). In contrast to the a c u t e treatment, chronic administration of 4 - e n e V P A to rats for 5 d a y s p r o d u c e d a n elevation of the mitochondrial G S H l e v e l s ( T a b l e 8).  This  apparently p a r a d o x i c a l result c a n be interpreted in terms of the stimulation of either the c y t o s o l i c e n z y m e s involved in the s y n t h e s i s of G S H ( R i c h m a n a n d M e i s t e r , 1975) or the transport s y s t e m carrying G S H into mitochondria, or both, in r e s p o n s e to the earlier depletion of cytosolic a n d mitochondrial G S H p o o l s by electrophilic metabolites of 4ene V P A .  S u c h a p h e n o m e n o n is not u n u s u a l for G S H - d e p l e t i n g a g e n t s . It w a s  reported that after administration of either 1,2-dichloropropane ( T r e v i s a n et a l . , 1989)  225  or 1,3-bis(2-chloroethyl)-1-nitrosourea (Smith, A . C . a n d B o y d , 1984) to rats a n initial depletion led to a later r e b o u n d i n c r e a s e in hepatic G S H .  A n i n c r e a s e in the  c o n c e n t r a t i o n of a c e t a m i n o p h e n w a s s e e n to s i m u l t a n e o u s l y e l e v a t e the s y n t h e s i s rate of G S H in isolated rat h e p a t o c y t e s (Dalhoff a n d P o u l s e n , 1992). T h e hepatotoxicity of 4 - e n e V P A m a y a l s o be a s s o c i a t e d with a n a c c u m u l a t i o n of G S S G in mitochondria, s i n c e a n inhibition of mitochondrial G R activity w a s o b s e r v e d in 4 - e n e V P A treated rats (Table 8). N o export of G S S G from the m i t o c h o n d r i a h a s b e e n d e t e c t e d , the o x i d i z e d thiol apparently b e i n g r e d u c e d in situ by G R o n c e it is formed (Olafsdottir a n d R e e d , 1988). A n a c c u m u l a t i o n of G S S G shifts the mitochondrial redox state a n d a d d s to the oxidative s t r e s s of the cell ( R e e d , 1990). T h i s m e c h a n i s m m a y in fact contribute to the cell toxicity o b s e r v e d for 4 - e n e V P A . T h e administration of ocfluoro-4-ene V P A did not p r o d u c e any statistically significant inhibitory effect o n the G R activity, w h i c h is a g a i n in a c c o r d with the o b s e r v e d n o n h e p a t o t o x i c properties of the compound.  Further s t u d i e s exploring the m e c h a n i s m of 4 - e n e V P A i n d u c e d inhibition  of G R are w a r r a n t e d . The  strongest argument against the h y p o t h e s i s of toxic metabolite(s)  being  r e s p o n s i b l e for V P A hepatotoxicity is that the toxicity is not correlated with the s e r u m c o n c e n t r a t i o n s of either 4 - e n e V P A or (E)-2,4-diene V P A in h u m a n s ( S i e m e s et a l . , 1993) a n d in a n i m a l s ( L o s c h e r et a l . , 1993).  T h e predication of s u c h a relationship  b e t w e e n drug metabolites a n d toxic effects is not n e c e s s a r i l y to be e x p e c t e d .  The  biotransformation of V P A a n d its metabolites entails two p h a s e I p a t h w a y s , n a m e l y mitochondrial p-oxidation a n d m i c r o s o m a l P 4 5 0 c a t a l y z e d oxidation ( S c h e m e 1.6, B)  226  (Baillie a n d Rettenmeier, 1989). pathway. VPA  T h e putative toxic metabolites m a y a r i s e from either  M i c r o s o m a l d e h y d r o g e n a t i o n of V P A a n d ( E ) - 2 - e n e V P A g i v e s rise to 4 - e n e  (Rettie et a l . , 1987) a n d (E)-2,4-diene V P A ( K a s s a h u n a n d B a i l l i e , 1993),  respectively. Mitochondrial p-oxidation of 4 - e n e V P A a l s o results in ( E ) - 2 , 4 - d i e n e V P A (Baillie a n d Rettenmeier, 1989), the difference b e i n g that the reactive intermediates such  as  (E)-2,4-diene  VPA-CoA  that  are  generated  within  mitochondria  may  immediately attack their targets to form covalent b o n d s . O n the other h a n d , the (E)-2,4d i e n e V P A arising from the m i c r o s o m a l oxidation of ( E ) - 2 - e n e V P A c o u l d h a v e l e s s impact o n hepatotoxicity d u e to its localization in the cytosolic compartment ( K a s s a h u n et a l . , 1994) ( S c h e m e 1.6, B). Experimentally, to differentiate in vivo w h e t h e r (E)-2,4d i e n e V P A is formed in m i c r o s o m e s from (E)-2-ene V P A or in m i t o c h o n d r i a from 4 - e n e V P A is very difficult to a c h i e v e w h e n both of the p r e c u r s o r s are present. C o n s e q u e n t l y , the toxic e v e n t s are unlikely to be anticipated by the s e r u m c o n c e n t r a t i o n s of either 4e n e V P A or (E)-2,4-diene V P A . It might e v e n be a r g u e d that it is the attack of reactive s p e c i e s with their targets that is important to the e x p r e s s i o n of toxicity.  Thus, reduced  s e r u m l e v e l s of 4 - e n e V P A or (E)-2,4-diene V P A c o u l d in fact be a s s o c i a t e d with more s e v e r e toxic c o n s e q u e n c e s . It is worthwhile here to mention o n e difference b e t w e e n our o b s e r v a t i o n s a n d that reported recently by L o s c h e r et a l . ( L o s c h e r et a l . , 1993) regarding 4 - e n e V P A i n d u c e d m i c r o v e s i c u l a r s t e a t o s i s in rats.  D o s e s of 4 - e n e V P A w e r e the s a m e in both  e x p e r i m e n t s e x c e p t that rats w e r e treated for 8 d a y s in the reported work, 3 d a y s longer than the protocol for our experiment.  L o s c h e r et a l . o b s e r v e d m i c r o v e s i c u l a r s t e a t o s i s  227  in l e s s than 5 0 % of the treated a n i m a l s with only 2 0 % h a v i n g a 4+ s c o r e ( L o s c h e r et al., 1993). T h i s contrasts with 1 0 0 % of the a n i m a l s being affected with a s c o r e greater than 4+ in this study a n d in the earlier work by K e s t e r s o n et a l . ( K e s t e r s o n et a l . , 1984) w h i c h w a s a l s o a five day study in vivo of 4 - e n e V P A hepatotoxicity.  T h e r e a s o n s for  t h e s e o b s e r v e d differences in experimental o u t c o m e are not readily apparent. B e c a u s e fluorine is a strong electron-withdrawing substituent, the introduction of fluorine into 4 - e n e V P A a n d 4 - P A will c h a n g e the p h y s i c a l c h e m i c a l properties of t h e s e compounds.  It  is p o s s i b l e that the  fluorinated  compounds  might  have  unique  p h a r m a c o k i n e t i c profiles that c o u l d lead to differences in the e x p r e s s i o n of toxicity. T h i s possibility h a s b e e n ruled out in our comparative p h a r m a c o k i n e t i c s t u d i e s (Section 4.6). S e v e r a l V P A derivatives, including V P A a n d (E)-2-ene V P A , h a v e b e e n s h o w n to inhibit fatty a c i d p-oxidation in rat liver mitochondria at levels c o m p a r a b l e to that of 4e n e V P A ( P o n c h a u t et a l . , 1992).  Y e t , V P A a n d ( E ) - 2 - e n e V P A are k n o w n to induce  m u c h l e s s m i c r o v e s i c u l a r s t e a t o s i s than 4 - e n e V P A in experimental a n i m a l s ( K e s t e r s o n et a l . , 1984).  S i n c e r e d u c e d utilization of fatty a c i d s in mitochondria is o n e of s e v e r a l  p o s s i b l e m e c h a n i s m s leading to a fatty liver, it may be c o n c l u d e d that a d e m o n s t r a t e d in vitro inhibition of p-oxidation d o e s not reflect the total picture of V P A hepatotoxicity. A l t h o u g h w e w e r e readily a b l e to p r o d u c e m i c r o v e s i c u l a r s t e a t o s i s in rat liver with 4e n e V P A treatment, n e c r o s i s , w h i c h is s e c o n d to s t e a t o s i s a s a c o m m o n l e s i o n s e e n in V P A hepatotoxicity  in h u m a n s (Zimmerman a n d Ishak, 1982), c o u l d not b e clearly  e s t a b l i s h e d . A fatty liver d o e s not n e c e s s a r i l y l e a d to the death of h e p a t o c y t e s a n d the  228  liver m a y still function a s normal ( P l a a a n d Hewitt, 1982).  Further investigations into  the m e c h a n i s m s of V P A hepatotoxicity m a y reveal the intricacies of t h e s e o b s e r v a t i o n s .  4.6. Comparative Pharmacokinetic Studies of 4-Ene VPA and cc-Fluoro-4-ene VPA in Rats  W e h a v e p r o p o s e d that oc-fluorination s h o u l d prevent the biotransformation of V P A a n d V P A metabolites through the p-oxidation pathway, a n d therefore eliminate the drug-related toxicity ( S e c t i o n s 1.3 a n d 4.5). S u b s e q u e n t toxicological s t u d i e s indicated that a - f l u o r o - 4 - e n e V P A w a s i n d e e d non-toxic in terms of l a c k i n g the toxic m a r k e r that is a s s o c i a t e d with 4 - e n e V P A , n a m e l y hepatic m i c r o v e s i c u l a r s t e a t o s i s , in the liver of a fluoro-4-ene V P A treated rats ( S e c t i o n s 3.6 a n d 4.5). T h i s investigation w a s d e s i g n e d to e x a m i n e the p h a r m a c o k i n e t i c a n d t i s s u e distribution properties of 4 - e n e V P A a n d a fluoro-4-ene V P A . T h e lipophilicities of 4 - e n e V P A a n d tx-fluoro-4-ene V P A w e r e d e t e r m i n e d to b e c o m p a r a b l e b a s e d o n the m e a s u r e m e n t s of H P L C c a p a c i t y factors.  Although 4-ene  V P A h a s a higher p K , both c o m p o u n d s a r e likely to b e present almost e x c l u s i v e l y in a  their i o n i z e d forms at p h y s i o l o g i c a l p H . T h e substitution of the a - h y d r o g e n in 4 - e n e VPA  with  a fluorine  atom  should  result  in  little  steric  effect  (Welch,  1990).  C o n s e q u e n t l y , the disposition of 4 - e n e V P A a n d oc-fluoro-4-ene V P A in rat livers w a s found to b e similar at 1 h post d o s e , a n d the s e r u m concentration-time profiles of the two d r u g s w e r e o b s e r v e d to r e s e m b l e o n e another during the initial 2 0 0 min following  229  the d o s e (Figure 3.34 a n d 3.35). W e h a d o b s e r v e d that hepatic mitochondrial G S H in 4 - e n e V P A , but not cc-fluoro-4-ene V P A , treated rats w a s d e c r e a s e d in the period of 0.75 - 3.0 h after giving the s a m e d o s e s a s in the present study (Section 3.6.2).  Our  p h a r m a c o k i n e t i c d a t a s u g g e s t that the distinction in depleting G S H w a s very unlikely to be a s s o c i a t e d with the different absorption a n d distribution properties of the two d r u g s . T h e apparent enterohepatic circulation of 4 - e n e V P A a n d ( E ) - 2 , 4 - d i e n e V P A c o u l d prolong the e x p o s u r e of liver towards t h e s e two toxic c o m p o u n d s a n d thus i n c r e a s e the risk of hepatic injury. VPA  analogues  is  due  largely  It w a s s u g g e s t e d that enterohepatic circulation of to  their  extensive  glucuronidation,  that  is,  the  g l u c u r o n i d e s of V P A , 4 - e n e V P A or (E)-2,4-diene V P A e x c r e t e d v i a the bile into the gastrointestinal tract are h y d r o l y z e d by p-glucuronidase, a n e n z y m e p r o d u c e d by gut b a c t e r i a ( B u e h l e r et a l . , 1 9 5 1 ; C l a r k et a l . , 1969), a n d the resultant parent c o m p o u n d s re-absorbed  into  the  systemic  circulation  (Singh  et  al.,  1988).  Because  of  enterohepatic circulation, a two-compartment m o d e l with a time-lag w a s u s e d to obtain the best-fit for the s e r u m drug concentration-time profile of 4 - e n e V P A . T h e time-lag pharmacokinetic m o d e l is a modified v e r s i o n of that p r o p o s e d by S t e i m e r et a l . (Steimer et a l . , 1982) for drugs w h i c h are subjected to e n t e r o h e p a t i c circulation, a n d w a s u s e d previously to fit the p l a s m a d a t a points of V P A a n a l o g u e s d o s e d i.v. to rats (Singh et a l . , 1988; 1990). T h e micro-rate c o n s t a n t s g e n e r a t e d from the m o d e l s u g g e s t that the net outflow of 4 - e n e V P A from the s y s t e m i c compartment to the peripheral compartment w a s attenuated only after a significant a c c u m u l a t i o n of the drug in the latter compartment b e c a u s e k-|2 w a s greater than k £ i . W h e n the net influx  230  from the peripheral compartment to s y s t e m i c circulation b e c a m e positive, a s e c o n d s e r u m p e a k concentration w o u l d start to e m e r g e . T h i s p r o c e s s s h o u l d be d e p e n d e n t o n 1) the f r e q u e n c y a n d v o l u m e of bile s e c r e t i o n ; 2) the activity of ^ - g l u c u r o n i d a s e e n z y m e s t o w a r d s the conjugate a n d 3) the absorption efficiency of the d e c o n j u g a t e d drug through the gastrointestinal wall. A p i e c e of e v i d e n c e that indirectly supports the h y p o t h e s i s that enterohepatic circulation of 4 - e n e V P A is the result of the glucuronidation p r o c e s s w a s o b t a i n e d from the metabolic a n d disposition s t u d i e s of a-fluoro-4-ene V P A in rats.  This compound  w a s found to form little g l u c u r o n i d e a n d w a s not o b s e r v e d to be r e - a b s o r b e d from the gut.  A s a result, the s e r u m concentration-time profile of a - f l u o r o - 4 - e n e V P A c o u l d  s i m p l y be s i m u l a t e d u s i n g a s i n g l e compartment m o d e l . Notably, the m o d e l g e n e r a t e d a micro-elimination rate constant  Q for oc-fluoro-4-ene V P A that w a s 0.67-fold that of  4 - e n e V P A , w h i c h w a s identical to the ratio of their apparent elimination c o n s t a n t s , i.e. 0.67. T h e (3-oxidative metabolite (E)-2,4-diene V P A w a s d e t e c t e d only in the s e r u m of 4 - e n e V P A treated rats, with the p e a k concentration of this toxic d i e n e o c c u r r i n g at 60 min following the d o s e .  P r e v i o u s l y , it w a s s h o w n in the rat that depletion of hepatic  mitochondrial G S H by 4 - e n e V P A w a s detected at 4 5 min after the d o s e (Section 3.6.2).  T h u s , a s l o w a c c u m u l a t i o n of (E)-2,4-diene V P A is temporally c o r r e l a t e d with  the o b s e r v e d d e c r e a s e in mitochondrial G S H , in a c c o r d a n c e with the a s s u m p t i o n that s u c h a depletion of G S H is d u e largely to the conjugation of G S H with (E)-2,4-diene V P A d e r i v e d from 4 - e n e V P A m e t a b o l i s m in mitochondria (Section 4.6).  231  D e s p i t e the apparent elimination rate constant, K, of 4 - e n e V P A b e i n g greater than  that of  comparable,  a-fluoro-4-ene largely  V P A , their  total c l e a r a n c e s  b e c a u s e enterohepatic  cycling  of  {CLj ) p  4-ene  a p p e a r e d to  VPA  be  contributed  significantly to the A U C (Table 11). T h e s e r u m p e a k concentration a n d protein binding of 4 - e n e V P A a n d a-fluoro-4-ene V P A w e r e o b s e r v e d to be similar ( T a b l e 11 a n d F i g u r e 3.33), a g a i n s u g g e s t i n g that contributions from the p h a r m a c o k i n e t i c properties of the two d r u g s to their biological effects would be fundamentally similar. C o n j u g a t i o n of xenobiotic carboxylic a c i d s with e n d o g e n o u s a m i n o a c i d s is b e l i e v e d to be initiated through the formation of the c o r r e s p o n d i n g a c i d C o A thioesters, followed by a c y l transfer to the a m i n o a c i d s (Hutt a n d C a l d w e l l , 1990).  The second  step of the conjugation reaction is known to be c a t a l y z e d by a c y l - C o A : a m i n o a c i d Na c y l t r a n s f e r a s e (Hutt a n d C a l d w e l l , 1990).  F o r e x a m p l e , a c y l - C o A : L-glutamine N-  a c y l t r a n s f e r a s e w a s isolated from liver mitochondria of the R h e s u s M o n k e y a n d human ( W e b s t e r et a l . , 1976). It w a s s h o w n that metabolism of V P A in rats c o u l d result in a g l y c i n e conjugate of the drug but the amount formed w a s l e s s than 1% of the d o s e ( G r a n n e m a n et a l . , 1984b).  oc-Fluorinated V P A a n a l o g u e s , o n the other h a n d ,  underwent e x t e n s i v e glutamine conjugation in mice (Section 3.8) a n d in rats.  The  urinary glutamine conjugate in rats w a s found to a c c o u n t for 2 2 % of oc-fluoro-4-ene V P A d o s e . T h e availability of the C o A thioester of a-fluoro-4-ene V P A b e c a u s e of the block in the (3-oxidation pathway is likely the main factor in the shift of the m e t a b o l i s m t o w a r d s the a m i n o a c i d conjugate, p r e s u m a b l y c a t a l y z e d by o n e of the mitochondrial a c y l - C o A : A/-acyltransferase e n z y m e s . T h e detection of biliary a - f l u o r o - 4 - e n e V P A - G I n  232  is c o n s i d e r e d to be indicative of the conjugation occurring in the liver, a n d this c o n c l u s i o n is s u p p o r t e d by a n in vitro study in w h i c h the glutamine conjugate of a fluoro-4-ene V P A w a s detected in rat hepatocyte incubations ( u n p u b l i s h e d data).  In  this respect, the mitochondrial C o A pool w a s quite p o s s i b l y perturbed following a d o s e of oc-fluoro-4-ene V P A , yet no hepatotoxicity w a s detected (Section 3.6.1).  T h i s result  w o u l d a r g u e against the h y p o t h e s i s that V P A hepatotoxicity is d u e to V P A i n d u c e d s e q u e s t r a t i o n of mitochondrial C o A (Turnbull et a l . , 1 9 8 3 ; Harris, R. A . et a l . , 1991). Further investigation is n e e d e d to clarify the effect of a - f l u o r o - 4 - e n e V P A o n hepatic C o A pools. A n o t h e r r e m a r k a b l e difference w a s o b s e r v e d in the m e t a b o l i s m of 4 - e n e V P A a n d a - f l u o r o - 4 - e n e V P A to form N A C conjugates, a n indicator of the in v i v o conjugation with G S H . T h r o u g h mitochondrial p-oxidation, 4 - e n e V P A , in the C o A thioester form, w a s c o n v e r t e d to (E)-2,4-diene V P A - C o A w h i c h reacted with G S H a n d w a s eventually e x c r e t e d in the urine a s 5 - N A C - 3 - e n e V P A ( K a s s a h u n et a l . , 1994). T h e (E)-2,4-diene V P A d e r i v e d from 4 - e n e V P A c o u l d a l s o react with G S H v i a a glucuronidation mediated pathway, a n d the urinary metabolite a s s o c i a t e d with s u c h a p r o c e s s w a s 5 - N A C - 3 - e n e V P A - g l u c u r o n i d e (Section 3.3.2). T h e m i c r o s o m a l P 4 5 0 c a t a l y z e d e p o x i d a t i o n of 4 - e n e V P A resulted in 4 , 5 - e p o x y V P A which further conjugated with G S H a n d w a s d e g r a d e d through the mercapturic a c i d pathway to give 5 - N A C - 4 - h y d r o x y V P A lactone in the urine ( K a s s a h u n et a l . , 1994). T h e s u m of the excreted N A C c o n j u g a t e s of 4 - e n e V P A w a s ~ 3 % of the d o s e .  233  S i m i l a r to 4 - e n e V P A biotransformation in e n d o p l a s m i c reticulum, oc-fluoro-4-ene V P A w a s c o n v e r t e d to the c o r r e s p o n d i n g 4 , 5 - e p o x i d e w h i c h reacted with G S H . T h e degraded  product  of the  G S H conjugate,  namely 5-NAC-2-fluoro-4-hydroxy V P A  lactone, w a s identified in the urine of rats treated with cc-fluoro-4-ene V P A . T h e amount of urinary 5 - N A C - 2 - f l u o r o - 4 - h y d r o x y V P A lactone, however, w a s only o n e tenth of its non-fluorinated counterpart p r o d u c e d in 4 - e n e V P A treated rats.  Additionally, neither  ( E ) - 2 , 4 - d i e n e V P A nor the N A C conjugate of (E)-2,4-diene V P A w a s d e t e c t e d in rats a d m i n i s t e r e d a-fluoro-4-ene V P A . T h u s , t h e s e d a t a a p p e a r to s u g g e s t that not only did the a-fluorination avert p-oxidation but a l s o d i m i n i s h e d the P 4 5 0 c a t a l y z e d epoxidation of the terminal d o u b l e b o n d . T h e electron-withdrawing ability of the fluorine substituent c o u l d a c c o u n t for this apparent r e s i s t a n c e of oc-fluoro-4-ene V P A t o w a r d s oxidative m e t a b o l i s m , b e c a u s e s u c h a n inductive effect w o u l d r e d u c e the stability of a putative cation radical intermediate which is generally b e l i e v e d to be involved in the  P450  m e d i a t e d epoxidation (Ortiz d e M o n t e l l a n o , 1989). W h i l e this h y p o t h e s i s r e m a i n s to be clarified, oc-fluoro-4-ene V P A apparently h a s little effect on hepatic G S H p o o l s a n d u n d e r g o e s l e s s p h a s e I metabolism than 4 - e n e V P A in rats. T h e latter w a s reflected in the r e c o v e r y of intact drug in rat urine.  Urinary a-fluoro-4-ene V P A w a s d e t e r m i n e d to  be 3 8 . 6 % of the d o s e , w h e r e a s urinary 4 - e n e V P A a c c o u n t e d for only 3 % of the d o s e . T a k e n together, the d a t a indicate that differences b e t w e e n a - f l u o r o - 4 - e n e V P A a n d 4e n e V P A r e s i d e in m e t a b o l i s m rather than absorption a n d disposition ( S c h e m e 4.5).  234  No toxicity  COOH Glucuronidation  COSCoA CoA  P-Oxidation  4  (a-Fluoro-4-ene VPA)  (a-Fluoro-4-ene VPA CoA thioester)  Glutamine conjugate  (2-Fluoro-4,5-epoxy VPA) GSH  NAC (2-Fluoro-5-GS-4hydroxy VPA lactone)  (2-Fluoro-5-NAC-4hydroxy VPA lactone)  S c h e m e 4 . 5 . M e t a b o l i s m of a-fluoro-4-ene V P A . Interruption in the p-oxidation p a t h w a y p r e v e n t e d hepatotoxicity w h i c h is a s s o c i a t e d with 4 - e n e V P A ( S e c t i o n 3.6.1).  235  4.7. Comparative Pharmacokinetic, Pharmacodynamic and Metabolic Studies of VPA and a-Fluoro VPA in Mice  B e c a u s e a-fluoro-4-ene V P A w a s found to b e non-toxic a s d e t e r m i n e d by the a b s e n c e of hepatic m i c r o v e s i c u l a r s t e a t o s i s in rats treated  c h r o n i c a l l y with  this  c o m p o u n d , the a-fluorinated V P A a n a l o g u e w a s thus s y n t h e s i z e d for e v a l u a t i o n of its anticonvulsant activity in m i c e . T h i s drug, however, w a s c h a r a c t e r i z e d by a s l o w onset of the anticonvulsant activity against P T Z - i n d u c e d s e i z u r e s , a n d this d e l a y e d effect w a s d e t e r m i n e d to b e d u e entirely to a s l o w e r uptake of a-fluoro V P A b y the brain a c c o r d i n g to the present p h a r m a c o k i n e t i c studies. T h e brain is efficiently s e p a r a t e d from the b l o o d by the b l o o d - b r a i n  barrier  ( B B B ) , w h i c h is k n o w n to h a v e s e v e r a l transport s y s t e m s regulating the p a s s a g e of e n d o g e n o u s a n d e x o g e n o u s c o m p o u n d s ( J e z e q u e l , S . G . , 1 9 9 2 , S p e c t o r , 1990). O n e of t h e s e is the m o n o - c a r b o x y l i c a c i d ( M C A ) carrier s y s t e m at the c e r e b r a l c a p i l l a r i e s w h i c h transports m o n o - c a r b o x y l i c a c i d s into the brain. It h a s b e e n r e c o g n i z e d that V P A c a n c r o s s the B B B through the M C A carrier s y s t e m at p h y s i o l o g i c a l p H ( T e r a s a k i et a l . 1991). O n the other h a n d , the relatively low brain concentration of V P A , a s c o m p a r e d to other anticonvulsant drugs s u c h a s phenytoin or p h e n o b a r b i t a l , led to investigations c o n c l u d i n g that the transport of V P A w a s asymmetric s u c h that the brain-to-blood efflux e x c e e d e d the blood-to-brain influx (Cornford et a l . , 1985).  R e c e n t d a t a s u g g e s t s that  p r o b e n e c i d - s e n s i t i v e a n i o n transporters a r e involved in the c l e a r a n c e of V P A from the C N S ( A d k i s o n et a l . 1994).  In a pharmacokinetic study, V P A w a s d e m o n s t r a t e d to  236  enter the C S F rapidly a n d l e a v e at a rate parallel to that d i s a p p e a r i n g from the p l a s m a (Levy,  1980)  In a c c o r d a n c e with this  finding,  it w a s o b s e r v e d in the  present  investigation that the arrival of p e a k V P A c o n c e n t r a t i o n s in the s e r u m a n d brain a p p e a r e d within 15 min of the d o s e a n d the apparent elimination rate c o n s t a n t s for t h e s e c o m p a r t m e n t s w e r e c o m p a r a b l e to e a c h other. T h e substitution of the a - h y d r o g e n in V P A with a fluorine atom s h o u l d a l s o result in little steric effect ( W e l c h , 1990). T h e lipophilicities of V P A a n d a-fluoro V P A were s h o w n to be c o m p a r a b l e . A l t h o u g h V P A h a s a higher p K , both c o m p o u n d s s h o u l d be a  present almost e x c l u s i v e l y in their i o n i z e d forms at p h y s i o l o g i c a l p H . If a-fluoro V P A is transported into the brain by a carrier-mediated s y s t e m similar to that r e s p o n s i b l e for V P A transportation, the s l o w e r a c c e s s of a-fluoro V P A into the brain c o u l d thus be d u e to either s e r u m drug-protein binding w h i c h w o u l d d e c r e a s e free drug concentration a v a i l a b l e for the transportation or a low affinity of the drug towards the active transport carriers. T h e results d e r i v e d from the p h a r m a c o k i n e t i c c a l c u l a t i o n s indicated that k-|2. a m e a s u r e of blood-to-brain influx, w a s s m a l l e r than k -|, a n index of brain-to-blood 2  efflux, implying that a-fluoro V P A , like V P A , might a l s o be a s y m m e t r i c a l l y transported a c r o s s the B B B .  S u c h a n asymmetric transport w a s further reflected in the lower  b r a i n / s e r u m drug concentration ratio of a-fluoro V P A than that of V P A . U n l i k e V P A , for w h i c h the brain/serum concentration ratio i n c r e a s e s rapidly after the d o s e followed by a d e c r e a s e with time ( H a m m o n d et a l . , 1982), the ratio for a-fluoro V P A w a s o b s e r v e d to reach a plateau (Section 3.8.2).  T h e s e results g i v e rise to the  s p e c u l a t i o n o n whether p a s s i v e diffusion is involved in transport of the drug into the  237  brain.  Finally, it s h o u l d be noted that the low brain/serum concentration ratio of a -  fluoro V P A , like its s l o w uptake by the brain, c o u l d a l s o be u n d e r the influence of s e r u m protein binding.  Attempts to c h a r a c t e r i z e the s e r u m protein binding properties of oc-  fluoro V P A w e r e not f e a s i b l e at this time owing to the limited availability of m o u s e b l o o d . Further investigations are d e e m e d n e c e s s a r y . S e r u m drug-protein binding m a y a l s o be partially r e s p o n s i b l e for the s l o w e r elimination of a-fluoro V P A from the g e n e r a l circulation. W h i l e the a p p a r e n t elimination of a-fluoro V P A from the brain paralleled its d i s a p p e a r a n c e from the s e r u m (Figure 3.37, A ) , the p h a r m a c o k i n e t i c m o d e l - g e n e r a t e d micro-constant k £ i w a s greater than the s e r u m elimination constant k-| Q, s u g g e s t i n g the c l e a r a n c e of the drug from the brain w a s limited by the c l e a r a n c e from the g e n e r a l circulation. T h e fluorinated V P A w a s o b s e r v e d to undergo very little glucuronidation a n d not to be a substrate of the p-oxidation e n z y m e s in rats ( S e c t i o n 3.6.3), w h e r e a s V P A is k n o w n to be e x t e n s i v e l y m e t a b o l i z e d in h u m a n a n d laboratory a n i m a l s (Baillie a n d R e t t e n m e i e r , 1989).  In this study, the primary metabolite of p-oxidation, ( E ) - 2 - e n e  V P A , w a s detected in the s e r u m a n d urine of m i c e a d m i n i s t e r e d V P A but not in a-fluoro V P A treated a n i m a l s . In contrast to the extensive glucuronidation of V P A , w h i c h w o u l d facilitate the c l e a r a n c e of the drug, little a-fluoro V P A g l u c u r o n i d e c o n j u g a t e w a s f o r m e d , indicating that the o b s e r v e d distinction b e t w e e n V P A a n d its a-fluorinated a n a l o g u e in both the p h a s e I a n d p h a s e II m e t a b o l i s m in m i c e m a y a c c o u n t for the a p p a r e n t difference in their elimination. T h e s l o w e r c l e a r a n c e of a-fluoro V P A from the  238  circulation  may  be a n a d v a n t a g e for the  drug  in terms  of the  duration  of  its  p h a r m a c o l o g i c a l activity. T h e formation of the L-glutamine conjugate by a-fluoro V P A is u n i q u e for a V P A a n a l o g u e . L i k e V P A , a-fluoro V P A c a n be similarly e x p e c t e d to form the c o r r e s p o n d i n g C o A thioester, while the further participation of the C o A thioester in p-oxidation is b l o c k e d by the fluorine substitutent.  T h i s interruption in the m e t a b o l i s m v i a p-oxidation  pathway w o u l d be r e a s o n a b l y a s s u m e d to shift the biotransformation of a-fluoro V P A C o A e s t e r to L-glutamine conjugation, b e c a u s e a c y l C o A s are g e n e r a l l y b e l i e v e d to be the p r e c u r s o r s of a c y l a m i n o a c i d c o n j u g a t e s (Hutt a n d C a l d w e l l , 1990). The  anticonvulsant activity of V P A is c o n s i d e r e d to be a s s o c i a t e d with its  capability of elevating s y n a p t o s o m a l G A B A levels in the brain ( L o s c h e r , 1993).  In  m i c e , 2 0 - 3 0 % i n c r e a s e s in the s y n a p t o s o m a l G A B A levels w e r e o b s e r v e d following the treatment with V P A at d o s e s of 125 - 2 9 0 mg/kg ( L o s c h e r , 1 9 8 1 ; L o s c h e r et a l . , 1984). C l i n i c a l r e l e v a n c e of the G A B A h y p o t h e s i s r e s i d e s in the o b s e r v a t i o n that C S F G A B A l e v e l s in 3 3 epileptic children on V P A therapy w e r e 2-fold higher than t h o s e of untreated patients a n d t h o s e treated by anticonvulsant d r u g s other than V P A ( L o s c h e r a n d S i e m e s , 1984). O n the other h a n d , a direct effect of V P A o n n e u r o n m e m b r a n e s h a s a l s o b e e n postulated.  V P A w a s d e m o n s t r a t e d to i n d u c e h y p e r p o l a r i z a t i o n in the  resting m e m b r a n e potential, p o s s i b l y d u e to i n c r e a s e d p o t a s s i u m c o n d u c t a n c e (Slater a n d J o h n s t o n , 1978; W a l d e n et a l , 1993). It w a s a l s o o b s e r v e d that V P A at therapeutic c o n c e n t r a t i o n s (6-200 u.M) c o u l d limit s u s t a i n e d repetitive firing in m o u s e central (spinal  239  c o r d a n d cortical) n e u r o n s without affecting postsynaptic G A B A r e s p o n s e s ( M c L e a n a n d M a c d o n a l d , 1986; M a c D o n a l d , 1988). In a g r e e m e n t with previously reported d a t a ( G a l e a n d l a d a r o l a , 1980), V P A w a s o b s e r v e d in this investigation to be a b l e to s i m u l t a n e o u s l y protect m i c e a g a i n s t P T Z i n d u c e d s e i z u r e s a n d i n c r e a s e m o u s e brain s y n a p t o s o m a l G A B A content after a n E D 5 0 d o s e ( T a b l e 14 a n d F i g u r e 3.38).  T h i s result m a y be v i e w e d a s a n o t h e r p i e c e of  e v i d e n c e w h i c h is in support of the h y p o t h e s i s that G A B A - e l e v a t i o n is o n e of the m e c h a n i s m s of V P A action. T h e G A B A - e l e v a t i o n m e c h a n i s m c o u l d a l s o be u s e d to interpret the o b s e r v e d a n t i c o n v u l s a n t activity of the cc-fluorinated a n a l o g u e . Administration of a-fluoro V P A to m i c e did i n c r e a s e synaptic G A B A levels, a n d the anticonvulsant effect of a-fluoro V P A w a s a s s o c i a t e d with the elevation of s y n a p t o s o m a l G A B A instead of the p e a k brain concentration of the drug at 2.08 mmol/kg d o s e s (Table 15). T h i s result m a y indicate that the capability of i n c r e a s i n g s y n a p t o s o m a l G A B A  is a factor in the  protection  p r o v i d e d by a-fluoro V P A a g a i n s t P T Z - i n d u c e d s e i z u r e s . A d e l a y in the e l e v a t i o n of s y n a p t i c G A B A c o m p a r e d with the brain drug concentration is p o s s i b l y d u e to either a s l o w distribution of a-fluoro V P A within the brain or to u n k n o w n regulation factors w h i c h either g o v e r n the interaction of the drug with a s yet u n d e t e r m i n e d r e c e p t o r s or limit the entry rate of the drug into the target c e l l s . H o w e v e r , argument c a n be m a d e a g a i n s t the G A B A h y p o t h e s i s on the b a s i s that despite a n elevation of brain s y n a p t o s o m a l G A B A no protection w a s p r o v i d e d to m i c e by a-fluoro V P A at d o s e s of 0 . 8 3 m m o l / k g .  These  p a r a d o x i c a l findings a p p e a r to imply that a different m e c h a n i s m of action or s e v e r a l  240  m e c h a n i s m s of action m a k e up the o b s e r v e d p h a r m a c o l o g i c a l effect of a-fluoro V P A . T h e b r o a d s p e c t r u m of anticonvulsant activity of V P A itself h a s b e e n s u g g e s t e d to be indicative of s e v e r a l m e c h a n i s m s of action being involved ( L o s c h e r , 1993).  241  5. Summary and Conclusions  T h i s r e s e a r c h p r o v i d e s a n u m b e r of unique insights into details regarding the biotransformation  of  V P A to  activated  metabolites.  A  part  of  this  work  was  a c c o m p l i s h e d by characterization a n d quantitation of drug related G S H conjugates. T h e detection of t h e s e thiol conjugates c a n s e r v e two p u r p o s e s : 1) provide structural identities for the "trapped" reactive intermediates g e n e r a t e d during the m e t a b o l i s m of V P A a n d V P A metabolites, a n d 2) provide a m e a s u r e for the in vivo reaction of G S H with potentially toxic metabolite(s) of V P A a n d thus indirectly e v a l u a t e e x p o s u r e of the b o d y to t h e s e reactive s p e c i e s . Firstly, L C / M S / M S a n d N M R e v i d e n c e clearly identified the G S H - g l u c u r o n i d e diconjugate of (E)-2,4-diene V P A in the bile of rats treated with the d i e n e .  W h e n 2,4-  d i e n e V P A g l u c u r o n i d e w a s i n c u b a t e d with G S H in the p r e s e n c e or a b s e n c e of G S T e n z y m e , the resultant di-conjugate w a s readily d e t e c t e d .  T h i s d i s c o v e r y pointed out  that the conjugation of G S H with (E)-2,4-diene V P A c a n be m e d i a t e d by its g l u c u r o n i d e ester, in addition to the putative C o A d e p e n d e n t pathway.  It is the first reported  i n s t a n c e in w h i c h a xenobiotic is activated by its g l u c u r o n i d e to further react with G S H via M i c h a e l addition. LC/MS/MS  evidence was  also  obtained  to  indicate  that  in  vivo  in  rats  d e g r a d a t i o n of the G S H conjugates of 4 - e n e V P A metabolites c a n o c c u r in the liver v i a the c y s t e i n y l g l y c i n e a n d c y s t e i n e conjugates to the c o r r e s p o n d i n g mercapturic a c i d s , a s o p p o s e d to a n inter-organ p r o c e s s which involves the kidney. T h i s o b s e r v a t i o n h a s  242  not b e e n reported for 4 - e n e V P A m e t a b o l i s m . T h e detection of the thiol c o n j u g a t e s in 4 - e n e V P A treated rats further confirmed that metabolic activation of 4 - e n e V P A l e a d s to electrophilic s p e c i e s which react with hepatic G S H . S u c h a reaction m a y p r o d u c e depletion of hepatic G S H r e s e r v e s . T h e results d e r i v e d from the in vitro incubation of the /V-acetylcysteamine ester of ( E ) - 2 , 4 - d i e n e V P A , a structural mimic of the c o r r e s p o n d i n g C o A thioester, a n d rat liver s u b c e l l u l a r fractions s u g g e s t that the addition of ( E ) - 2 , 4 - d i e n e V P A to G S H is c a t a l y z e d by hepatic G S T e n z y m e s with the esterified d i e n e b e i n g e s s e n t i a l .  The  catalytic effect o n the conjugation by G S T e n z y m e in c y t o s o l c a n be further e n h a n c e d by prior administration  of P B .  In view of the p o l y m o r p h i c  nature  of G S T , this  information is important b e c a u s e t h e s e e n z y m e s m a y actually be involved in promoting the depletion of hepatic G S H r e s e r v e s by V P A reactive metabolites. In addition to conjugation with hepatic G S H , the activated forms of (E)-2,4-diene V P A , n a m e l y the c o r r e s p o n d i n g g l u c u r o n i d e a n d thioester, w e r e both d e m o n s t r a t e d to be c a p a b l e of alkylating r e d u c e d oxytocin at the free c y s t e i n e r e s i d u e s .  A direct  modification of critical proteins by the d i e n e metabolite of V P A a p p e a r s p o s s i b l e . Further toxicological s t u d i e s c o n d u c t e d in vivo identified the critical role of poxidation in V P A a s s o c i a t e d liver injury. W i t h the aid of a - f l u o r o - 4 - e n e V P A acting a s a m e c h a n i s t i c probe, it w a s evident that the hepatotoxicity of 4 - e n e V P A , a n d p o s s i b l y V P A , requires metabolic activation of the drug in mitochondria.  T h e rationale for this  c l a i m is the fact that 4 - e n e V P A , but not a-fluoro-4-ene V P A , p r o d u c e d s e v e r 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 in rats.  T h e metabolite (E)-2,4-diene V P A a n d the N A C  243  conjugate of (E)-2,4-diene V P A were detected only in 4 - e n e V P A treated rats, the N A C conjugate b e i n g a n indication for the in vivo reaction of the d i e n e with G S H . Depletion of G S H a n d inhibition of G R in liver mitochondria w e r e a l s o s e l e c t i v e for 4 - e n e V P A . C o n s i s t e n t with the d a t a obtained from the metabolic s t u d i e s , t h e s e results s u g g e s t e d that a n impaired G S H redox s y s t e m c o u l d be a factor contributing  to the  drug  a s s o c i a t e d hepatotoxicity. T o solidify s u c h a c l a i m , s u b s e q u e n t p h a r m a c o k i n e t i c s t u d i e s in rats indicated that the s e r u m concentration-time  profiles of 4 - e n e V P A a n d oc-fluoro-4-ene  VPA  r e s e m b l e d o n e another during the time frame w h e r e i n differences w e r e o b s e r v e d for their effects o n mitochondrial G S H . T h e disposition in rat livers a n d s e r u m free drug c o n c e n t r a t i o n s w e r e a l s o c o m p a r a b l e for the two c o m p o u n d s .  T h u s , it is c o n c l u d e d  that the distinction b e t w e e n 4 - e n e V P A a n d its a-fluorinated a n a l o g u e to p r o d u c e liver toxicity  in  rats  is not  a s s o c i a t e d with any  major  differences  in  absorption  and  distribution. O u r results a p p e a r to s u g g e s t that further investigations of V P A a s s o c i a t e d hepatotoxicity will n e e d to f o c u s on the formation of reactive intermediate(s).  For  e x a m p l e , the toxicity s h o u l d correlate with the total amount of the e n d p r o d u c t s arising from reactive intermediates rather than with s e r u m drug or metabolite c o n c e n t r a t i o n s . F r o m a clinical point of view, caution must be e x e r c i s e d w h e n V P A is u s e d in G S H d e f i c i e n c y s t a t e s , w h i c h c a n be inherited, s u c h a s a d e f i c i e n c y in o n e of the e n z y m e s in the p a t h w a y s l e a d i n g to G S H production, or a n a c q u i r e d d e f i c i e n c y of G S H , s u c h a s t h o s e a s s o c i a t e d with HIV or hepatic cirrhosis.  244  T h e findings  a l s o s u g g e s t that  preventing p-oxidation to a reactive s p e c i e s m a y n e e d to be c o n s i d e r e d a s o n e of the criteria in the m o l e c u l a r d e s i g n of n e w V P A a n a l o g u e s . O n e s u c h c o m p o u n d is a-fluoro V P A w h i c h w a s determined to be inert to the poxidation pathway.  T h e drug is c h a r a c t e r i z e d by its s l o w a c c e s s into the brain.  But,  o n c e in the brain, a-fluoro V P A a p p e a r e d to persist, p o s s i b l y resulting from its s l o w elimination from the circulation.  T h e drug w a s c a p a b l e of s u p p r e s s i n g P T Z promoted  s e i z u r e s in m i c e at a n E D 5 0 d o s e of 1.70 mmol/kg. T h e a-fluoro a n a l o g u e of V P A h a s potential a s a new anticonvulsant, a n d c o u l d be free of the idiosyncratic hepatotoxicity that is a s s o c i a t e d with V P A .  245  6. References Abbott, F. S . a n d A c h e a m p o n g , A . A . : Quantitative structure-anticonvulsant activity relationships of valproic a c i d , related carboxylic a c i d s a n d tetrazoles. N e u r o p h a r m a c o l o g y 27: 2 8 7 - 2 9 4 , 1988. Abbott, F. 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