The identification, characterization and profiling of novel thiol and amino acid conjugates of valproic acid in humans and animals

Valproic acid (VPA) is an antiepileptic drug used for the management of generalized and absence seizures. It suffers from a rare but fatal hepatotoxicity side effect and its mechanism of action is not known. We demonstrated that the urinary thiol conjugates of (E)-2,4-diene VPA, a reactive intermediate arising from the further biotransformation of the metabolite 4-ene VPA, were significantly elevated in patients at high risk of developing the side effect. Therefore, these conjugates reflect a higher exposure towards the reactive intermediate of 4-ene VPA in high risk patients. First, we characterized the structure of NAC II as 5-A/-acetylcystein-S-yl-2-ene VPA by 1H NMR. Then, using GC/MS and LC/MS/MS, we confirmed the identification of NAC I (previously detected in humans) and NAC II in patients on VPA therapy (n=40). The profiling of NAC I and II in urine samples of patients on VPA alone (group 1) or VPA in combination with non-enzyme inducing drugs (group 2), showed that patients < 7.5 years old excreted significantly higher concentrations of the two conjugates compared to older patients (>7. 5 years) in both groups (p<0.05). Furthermore, patients on VPA polytherapy with enzyme inducing drugs (group 3 and > 7.5 years old), excreted significantly higher concentrations of NAC I and NAC II compared to patients in groups 1 and 2 and of similar age (p