Identification and quantitation of novel metabolites of amiodarone in plasma of treated patients

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Abstract

In mammals, mono-N-desethylamiodarone (MDEA) is the only known metabolite of amiodarone. Our previous experiments demonstrated that in vitro MDEA may be hydroxylated, N-dealkylated, and deaminated. In this report, we investigated the concentration of these microsomal metabolites in the plasma of patients receiving amiodarone. The presence of the hydroxy-amiodarone and deiodinated amiodarone was also additionally investigated. A high-performance liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (HPLC–APCI-MS/MS) quantitative assay using morpholine–amiodarone as internal standard was developed for measuring these metabolites in the range of 3–250 ng ml−1. In the concentration ranges 5–50 and 50–250 ng ml−1, the coefficients of variation of the measurements were less than 14 and 7%, respectively. The concentrations of investigated compounds in plasma of patients (n = 14) receiving amiodarone (0.2 g day−1, orally for >2 months) varied inter-individually and were 140.0 ± 85.2, 39.1 ± 20.8, and 26.2 ± 15.2 ng ml−1 for 3′OH-mono-N-desethylamiodarone, di-N-desethylamiodarone, and deaminated amiodarone, respectively. The concentrations of MDEA and amiodarone in these samples were 970 ± 347 and 11163 ± 435 ng ml−1, respectively. In contrast, the studied compounds were not detectable in plasma samples from eight patients receiving amiodarone intravenously. Qualitatively, in the plasma of patients receiving amiodarone orally, hydroxylated amiodarone was also positively detected by assaying the [M + H]+ ions at m/z 662, but the deiodo-metabolites of amiodarone were not detected using mass spectrometry. Thus, in humans, amiodarone and MDEA were biotransformed by dealkylation, hydroxylation, and deamination.

Introduction

Amiodarone, which was initially developed 40 years ago as an antianginal drug, is still considered as one of the most effective antiarrhythmic agents (Mason, 1987, Julian et al., 1997). However, amiodarone therapy is accompanied by a variety of severe adverse effects, including pulmonary toxicity (Heger et al., 1983, Marchlinski et al., 1982), hepatotoxicity (Rigas et al., 1986), and thyroid dysfunction (Dickstein et al., 1984). The incidence of undesired effects appears to be dose dependent (Heger et al., 1983, Hilleman et al., 1998). Since the metabolism of amiodarone is not fully documented, the relationship between undesired effects and amiodarone metabolite(s) is poorly understood. In the present research situation, it is known that, in mammals, amiodarone is dealkylated to mono-N-desethylamiodarone (MDEA) (Flanagan et al., 1982). This pharmacologically active metabolite was found in in vitro experiments (Ha et al., 1992, Trivier et al., 1993, Fabre et al., 1993). Recently, it was observed that in vitro MDEA may be further cleared by hydroxylation (Ha et al., 2001), dealkylation to di-N-desethylamiodarone (DDEA), and deamination to deaminated amiodarone (DAA) (Kozlik, 2003). Independently of this, it is well known that amiodarone and its derivatives are very lipophilic; they are largely stored in the organ tissues rather than abundantly present in blood circulation. Thus, investigation of amiodarone metabolites in organ tissues should be more informative; however, such a study is not possible in humans. An alternative for studying amiodarone metabolism in humans is to use a sensitive and selective analytical assay to investigate metabolites other than MDEA in the plasma of patients receiving amiodarone.

Section snippets

Chemicals

Amiodarone [2-n-butyl-3-(3,5-diiodo-4-β-diethylaminoethoxybenzoyl)benzofuran hydrochloride] hydrochloride and mono-N-desethylamiodarone [2-n-butyl-3-(3,5-diiodo-4-β-ethylaminoethoxybenzoyl)benzofuran] hydrochloride (MDEA·HCl) were a kind gift from Sanofi-Synthélabo Research Center, Montpellier, France. The amiodarone analogue 2-n-butyl-3-(3,5-diiodo-4-hydroxybenzoyl)benzofuran (B2) was synthesized in previous studies (Ha et al., 2000). Other reference compounds, such as deaminated amiodarone [2-

Qualitative and quantitative analysis of known amiodarone metabolites

Our primary investigations indicated that 90–95% of the three studied compounds — 3′OH-MDEA, DDE, and DAA — and the internal standard MOPAM may be extracted with isooctane:2-propanol (85:15). This mixture of organic solvents had been used previously for extracting amiodarone and its related compounds from biological samples at pH 5.4 (Heger et al., 1984). We optimized the extraction for our analytes and observed that at pH 7.4, the extraction efficacy of our studied compounds was quantitative,

Discussion

The main finding in this study was the positive detection in human plasma of three compounds, which have been observed in our previous in vitro experiments (Kozlik, 2003), using the HPLC–APCI-MS/MS assay. This cannot be the artifact of the experiments, because they were not detected in plasma of patients receiving amiodarone intravenously. Among the new metabolites, the concentration of 3′OH-MDEA was 140.0 ng ml−1 (range 37–357 ng ml−1) and corresponded to 12.0% (range 3.58–23.5%) of the plasma

Acknowledgement

The authors thank Aileen McAinsh, Ph.D., Nashville, TN, USA, for assistance in editing the manuscript.

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