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

University of British Columbia

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<0.05). There were no significant difference between the conjugates excreted by patients in group 3 and patients in groups 1 and 2 who were less than 7.5 years old. Therefore, age, dose, and polytherapy, the risk factors of VPA induced hepatotoxicity, were found to be associated with significantly higher concentrations of urinary NAC I and NAC II in patients on VPA therapy. Furthermore, we reported the identification and characterization of novel valproyl glutamate (VPA GLU) and valproyl glutamine (VPA GLN) in the urine, serum and CSF samples of humans on VPA therapy, rats and rabbits treated with VPA. VPA GLU is the first glutamate conjugate of a xenobiotic discovered in humans, rats and rabbits. In addition, we were able to identify valproyl glycine (VPA GLY) in the urine and serum of humans on VPA therapy. These conjugates were not associated with the risk factors of VPA induced hepatotoxicity. The detection of VPA GLU in human CSF and at a concentration higher than the corresponding serum concentration suggests that the mechanism of action of VPA can be partially expressed through the conjugation of VPA with the excitatory neurotransmitter glutamic acid itself. Since, valproyl glycinamide, the amide of VPA GLY, was found to possess antiepileptic activity and is currently undergoing clinical trials (Hadad and Baieler, 1995), the detection of VPA GLU and VPA GLN in human CSF implies that both conjugates could have antiepileptic or CNS activity. The detection of VPA GLU in rabbit CSF supports our human findings.

Thesis/Dissertation

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2009-06-19

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http://hdl.handle.net/2429/9469

Pharmaceutical Sciences

University of British Columbia

UBCV

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