Analytical power of LLE–HPLC–PDA–MS/MS in drug metabolism studies: Identification of new nabumetone metabolites

Highlights

• Missing link in nabumetone metabolic pathways was discovered using LLE–HPLC–PDA–MS/MS.

• New nabumetone(NB) metabolite was extracted from liver microsomal fractions (man,rat).

• 3-Hydroxy nabumetone (3HO-NB) was identified via (a)chiral LLE–HPLC–PDA–MS/MS analyses.

• 3HO-NB was synthesized and its metabolism was studied via microsomes and hepatocytes.

• In isolated hepatocytes, 3HO-NB was transformed to 6-MNA, the PD active NB metabolite.

Abstract

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date.

In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE–HPLC–PDA and achiral LLE–UHPLC–MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC–MS/MS experiments detected the presence of a structure corresponding to elemental composition C₁₅H₁₆O₃, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite.

To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE–HPLC–PDA–MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone → 3-hydroxy nabumetone → 6-MNA).

Introduction

The nonsteroidal anti-inflammatory prodrug nabumetone possesses only a weak cyclo-oxygenase 2 (COX-2) inhibitory activity [1]. The originality of the neutral nabumetone structure arises from a relatively low incidence of gastric irritancy, bleeding, ulcerations and even gastrointestinal perforations, which were often observed following a long-term use of various NSAID active arylalkane acids. Lacking the mentioned adverse effects, neutral nabumetone is well absorbed from the GIT, and transported in the liver, where it undergoes an extensive first-pass biotransformation. Oxidative cleavage of nabumetone side-chain leads to the main pharmacodynamically active metabolite, a very strong COX-2 inhibitor, 6-methoxy-2-naphthylacetic acid (6-MNA). Similar to other arylalkane acids, 6-MNA displays anti-inflammatory and analgesic effects. O-Desmethylation and carbonyl reduction have also been reported as additional metabolic conversions of nabumetone [2], [3]. Hydrophilic phase II nabumetone metabolites, namely acyl glucuronide from 6-MNA, ether glucuronides from O-desmethyl and phase I nabumetone metabolites with reduced carbonyl were found in the urine, bile and duodenal mucosa of minipigs as well as in human urine [4].

An overview of analytical approaches to the determination of nabumetone in pharmaceutical applications and nabumetone biotransformation products in bioanalytical studies has been provided in our previous communication [3]. Recent analytical methods for nabumetone determination based on voltammetric techniques [5], heavy atom-induced phosphorescence [6], spectrophotometric methods [7], TLC or HP TLC densitometry [8], [9] or HPLC [10], [11], [12], [13] have also been reported in the last decade. Characterization of the enzymes involved in the biotransformation of nabumetone to 6-MNA [14] and in O-desmethylation of 6-MNA to 6-HNA [15] has already been performed.

It is very surprising that no biotransformation intermediate(s) between nabumetone and 6-MNA have been discovered since 1984, when Haddock et al. published an overview of nabumetone metabolites for the first time [2]. The loss of two carbon atoms indicates an initial conversion of nabumetone to oxidative product(s), the identification of which could help to elucidate the detailed course of nabumetone conversion to 6-MNA.

Our attempt to find the missing link(s) in nabumetone → 6-MNA conversion commenced by in vitro evaluation of nabumetone biotransformation using human and rat liver subcellular fractions (microsomes and cytosol), and rat isolated hepatocytes. This xenobiochemical approach combined with LLE–HPLC–PDA–MS/MS enabled the detection and identification of new nabumetone metabolite(s).