Reactivity of atropaldehyde, a felbamate metabolite in human liver tissue in vitro

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Abstract

Antiepileptic therapy with a broad spectrum drug felbamate (FBM) has been limited due to reports of hepatotoxicity and aplastic anemia associated with its use. It was proposed that a bioactivation of FBM leading to formation of α,β-unsaturated aldehyde, atropaldehyde (ATPAL) could be responsible for toxicities associated with the parent drug. Other members of this class of compounds, acrolein and 4-hydroxynonenal (HNE), are known for their reactivity and toxicity. It has been proposed that the bioactivation of FBM to ATPAL proceeds though a more stable cyclized product, 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one (CCMF) whose formation has been shown recently. Aldehyde dehydrogenase (ALDH) and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes. This study examined effects of ATPAL and its precursor, CCMF on ALDH, GST and cell viability in liver, the target tissue for its metabolism and toxicity. A known toxin, HNE, which is also a substrate for ALDH and GST, was used for comparison. Interspecies difference in metabolism of FBM is well documented, therefore, human tissue was deemed most relevant and used for these studies. ATPAL inhibited ALDH and GST activities and led to a loss of hepatocyte viability. Several fold greater concentrations of CCMF were necessary to demonstrate a similar degree of ALDH inhibition or cytotoxicity as observed with ATPAL. This is consistent with CCMF requiring prior conversion to the more proximate toxin, ATPAL. GSH was shown to protect against ALDH inhibition by ATPAL. In this context, ALDH and GST are detoxifying pathways and their inhibition would lead to an accumulation of reactive species from FBM metabolism and/or metabolism of other endogenous or exogenous compounds and predisposing to or causing toxicity. Therefore, mechanisms of reactive aldehydes toxicity could include direct interaction with critical cellular macromolecules or indirect interference with cellular detoxification mechanisms.

Introduction

Felbamate (FBM) is a broad spectrum antiepileptic drug. It was approved in 1993 by the United States Food and Drug Administration for the treatment of several forms of epilepsy, including monotherapy and adjunctive therapy for partial and generalized seizures and Lennox–Gastaut syndrome and related disorders, for which there have been very few therapeutic options. After its release, several cases of severe idiosyncratic adverse reactions (aplastic anemia and hepatotoxicity) were reported in patients treated with FBM. Due to these potential risks [1], FBM is not indicated as first line treatment [2].

Although the etiology of FBM-induced toxicities remains unknown, strong evidence is emerging that these idiosyncratic untoward reactions may be a consequence of bioactivation of FBM to reactive metabolites [3], [4]. At the time of its introduction on the market, four metabolites of FBM were identified in humans [5] and experimental animals [6], [7]. These metabolites were, 2-(4-hydroxyphenyl)-1,3-propanediol dicarbamate (pOH-FBM); 2-hydroxy-2-phenyl-1,3-propanediol dicarbamate (2OH-FBM); 2-phenyl-1,3-propanediol monocarbamate (MCF), and 3-carbamoyl-2-phenylpropionic acid (CPPA)(Fig. 1). Subsequent to reports of idiosyncratic toxicities, mercapturate metabolites, N-acetyl-S-(2-phenylpropan-3-ol)-l-cysteine (Nac-alcohol) and N-acetyl-S-(2-phenylpropanoic acid)-l-cysteine (Nac-acid), were identified in human urine [8]. Presence of these mercapturates suggested reactive metabolite formation and led to a proposed metabolic scheme for FBM in Fig. 1, [8]. According to this scheme, reactive aldehydes are the intermediate metabolites and are likely responsible for the observed toxicities of FBM. A monodecarbamoylated metabolite of FBM, MCF is formed by as yet unidentified esterase(s), amidase(s), and/or carboxylesterase(s) and oxidized, probably by alcohol dehydrogenase(s). The resultant aldehyde, 3-carbamoyl-2-phenylpropionaldehyde can undergo reversible cyclization to CCMF [8], oxidation to CPPA (the major metabolite in humans in vivo [5]) by aldehyde dehydrogenase(s) (ALDH), or β-elimination (spontaneous or catalyzed) to ATPAL. ATPAL, an α,β-unsaturated aldehyde, is a potent electrophile that can undergo rapid conjugation with cellular nucleophiles, including glutathione, and is now thought to be the proximate toxic metabolite of FBM. The α,β-unsaturated aldehyde moiety is present in other reactive compounds, e.g. HNE and acrolein, and has been suggested to be responsible for their toxicities [9], [10]. CCMF, on the other hand, is relatively stable and could represent a metabolic depot and part of a mechanism of transport from the site(s) of its formation to distal regions where it can convert to the ultimate reactive metabolite ATPAL.

Marked interspecies differences were observed for metabolism of FBM [11], [12] (Fig. 1). In vivo in man, hydroxylated products (pOH-FBM and 2OH-FBM) of FBM represent only minor metabolites (∼10% of the total FBM dose) and majority of metabolism occurs by formation and oxidation of MCF. Conversely, the hydroxylated metabolites of FBM are major products in rat in vivo (∼62% of the total FBM dose) [12]. A comparison of FBM metabolism in rat and human has demonstrated a protective metabolism in rat relative to man. ATPAL formation is 6-fold greater in humans than rats when measured as excreted mercapturates. The difference is thought to result from differences in P450, esterase and ALDH activities [11]. In dog, metabolic profile is intermediate between that in man and rat [12].

It is important to select relevant species when conducting metabolic and toxicological studies. Our previous publication [3] confirmed the formation of the proposed aldehyde metabolites of FBM using human liver tissue in vitro (microsomes, S9 fractions, and liver slices). These data lend a strong support to the proposed intermediate toxic pathway of FBM metabolism. Since ALDH and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes, the present study was designed to investigate the effects of ATPAL and its precursor, CCMF, on ALDH, GST and cell viability in human liver tissue in vitro.

Section snippets

Materials

FBM, MCF, and CPPA were provided by Wallace Laboratories (Cranbury, NJ). CCMF and ATPAL were synthesized as described previously [4]. Atropic acid was synthesized, purified and characterized at University of Virginia (Department of Chemistry, Charlottesville, VA). Methanol, methyl-tert-butyl ether and acetonitrile were obtained from Burdick and Jackson (Muskegon, MI). EDTA (disodium salt), potassium phosphate, magnesium chloride, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium

Results

Inhibition of ALDH by the putative proximate toxin, ATPAL and its cyclized precursor, CCMF was studied in human S9 fractions, in the presence and absence of GSH and using indole-3-acetaldehyde as a prototype substrate. For comparison, effects of another α,β-unsaturated aldehyde, HNE were also evaluated. ATPAL and HNE exhibited inhibition at similar concentrations using human liver S9 fractions (Fig. 2). Both produced greater than 50% inhibition at 10 μM concentrations. CCMF was less effective

Discussion

Proposed metabolic scheme for FBM involving reactive metabolite pathway implicates formation of α,β-unsaturated aldehydes (Fig. 1). The end products of this reactive pathway, Nac-alcohol and Nac-acid, have been identified in urine from patients on FBM [8]. Trapping of the intermediate aldehyde metabolites in form of stable oxime derivatives demonstrated their formation by human liver tissue in vitro, S9 fractions, microsomes, and liver slices [3]. Subsequently, formation of these metabolites

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