A serious drawback in the use of the anticonvulsant agent valproic acid (VPA) is
the drug associated liver toxicity characterized by microvesicular steatosis frequently
accompanied by necrosis. The main objective of this thesis was to test the hypothesis
that the hepatotoxicity of VPA is due to the formation of reactive toxic metabolites.
Firstly, metabolic activation of VPA was investigated by detection and
characterization of drug-related thiol conjugates. Combined LC / MS / MS and NMR
evidence clearly identified 5-GS-3-ene VPA-glucuronide I in the bile of rats dosed with
(E)-2,4-diene VPA which is suspected to play a key role in VPA hepatotoxicity.
Sufficient on-line LC / MS / MS data were obtained to indicate the presence of the NACglucuronide
di-conjugate of (E)-2,4-diene VPA in both rat bile and urine. The amount
of biliary 5-GS-3-ene VPA-glucuronide I was 7-fold greater than 5-GS-3-ene VPA, the
sum of the two metabolites accounting for 6.6% of the dose. Incubation of 2,4-diene
VPA-glucuronide with GSH in the presence of glutathione S-transferase (GST) enzyme
led to the formation of the GSH-glucuronide di-conjugate. To the best of our
knowledge, this is the first recorded instance in which glucuronide formation activates a
drug to further conjugate with GSH via a Michael addition reaction.
In other experiments, LC / MS / MS analysis of bile samples collected from rats
dosed with 4-ene VPA, an analogue of the known hepatotoxicant 4-pentenoic acid
(4PA), indicated the presence of the GSH, cysteinylglycine, cysteine and NAC
conjugates of 4,5-epoxy VPA and (E)-2,4-diene VPA , respectively. Quantitatively, the
biliary thiol conjugates accounted for 5% of the dose. This observation is novel for 4-
ene VPA metabolism in terms of the degradation of GSH conjugates possibly occurring
within the liver as opposed to an inter-organ process which involves the kidney. The
GSH - and NAC-glucuronide di-conjugates of (E)-2,4-diene VPA were also identified as
metabolites with 5-GS-3-ene VPA-glucuronide I representing 0.03% of the 4-ene VPA
dose. Taken together, these data clearly indicate that reactive metabolites of VPA can
react with hepatic GSH via several different metabolic pathways, the subsequent
depletion of GSH having potential toxic consequences. Additionally, (E)-2,4-diene
VPA, in its esterified forms, was demonstrated to be capable of alkylating reduced
oxytocin at the free cysteine residues, implicating a direct modification of critical
proteins by the diene metabolite of VPA .
The role of GST in the conjugation of GSH with (E)-2,4-diene VPA was
investigated using rat liver subcellular fractions as the source of GST enzymes. The
GST mediated conjugation of GSH with (E)-2,4-diene VPA A/-acetylcysteamine
thioester, a structural mimic of the corresponding CoA thioester, resulted in two
isomeric products via either 5,6- or 1,6-addition, in agreement with in vivo observations.
Only the 1,6-addition product was found for the spontaneous reaction of GSH with the
unsaturated thioester (control). Quantitatively, GSH conjugates formed in the presence
of the cytosol and sonic disrupted mitoplasts were 23- and 2-fold that of control,
respectively. No reaction could be detected upon a mix of GSH with the free acid form
of (E)-2,4-diene VPA. The results indicate that GST enzymes enhance the addition of
GSH to (E)-2,4-diene VPA with the esterified diene being essential for the reaction.
To further examine the metabolic activation hypothesis, oc-fluoro-4-ene VPA
which was expected to be inert to p-oxidative metabolism was synthesized and its
effect on rat liver studied in comparison with 4-ene VPA . Following treatment of rats for
5 days, 4-ene VPA, but not a-fluoro-4-ene VPA, induced severe hepatic microvesicular
steatosis (> 8 5% affected hepatocytes) and alterations in mitochondria. Similar results
were obtained when 4-pentenoic acid and 2,2-difluoro-4-pentenoic acid were
compared. The p-oxidation product of 4-ene VPA, namely (E)-2,4-diene VPA , and the
^-acetylcysteine (NAC) conjugate of the diene could not be detected in rats
administered oc-fluoro-4-ene VPA . In a separate acute study, mitochondrial GSH was
determined to remain unchanged in rats treated with cc-fluoro-4-ene VPA but was
reduced to 68% of control in those administered 4-ene VPA . These data are consistent
with results derived from metabolic studies, suggesting that formation of a reactive
intermediate is a key step in the events leading to 4-ene VPA, and possibly VPA,
induced liver injury with depletion of mitochondrial GSH as one of the causative factors.
A subsequent investigation was carried out to compare 4-ene VPA and a-fluoro-
4-ene VPA for their pharmacokinetic and protein binding properties. The serum
concentration-time profiles of 4-ene VPA and a-fluoro-4-ene VPA were observed to
resemble one another during the initial 200 min within which differences were apparent
for the drug effects on mitochondrial GSH . The major phase II metabolites were the L-glutamine
conjugate for cc-fluoro-4-ene VPA and the glucuronide ester for 4-ene VPA .
The toxic metabolite (E)-2,4-diene VPA and its NAC conjugate were again detected
only in 4-ene VPA treated rats. Despite differences in metabolism, the disposition to rat
liver, the serum peak and free concentrations were comparable for 4-ene VPA and ocfluoro-
4-ene VPA. Thus, the apparent distinction between the two drugs in producing
liver toxicity in rats is unlikely to be associated with pharmacokinetic differences.
Finally, because of the apparent nonhepatotoxic property of a-fluoro-4-ene VPA ,
a-fluoro VPA was evaluated for anticonvulsant activity in mice. The ED50 of the drug
was determined to be 1.7 mmol/kg with the peak activity occurring at 45 - 60 min
following the dose, in contrast to 10 min for VPA . Subsequent kinetic studies revealed
that the brain uptake of a-fluoro VPA was slower, the peak brain concentration arriving
45 min later than in the serum, whereas the peak brain level of VPA coincided with the
peak serum level occurring within 15 min of the dose. On the other hand, a-fluoro VPA
appeared to persist in the general circulation, resulting in its apparent slow elimination
from the brain. a-Fluoro VPA was demonstrated to have anticonvulsant activity in the
pentamethylenetetrazole seizure test in mice and to be capable of increasing brain
synaptosomal GABA, although the connection between these two events remains to be
clarified. These results suggest that a-fluoro VPA has potential as a new
anticonvulsant drug. / Pharmaceutical Sciences, Faculty of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6204 |
| Date | 05 1900 |
| Creators | Tang, Wei |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 14286176 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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