Gemfibrozil and its oxidative metabolites: quantification of aglycones, acyl glucuronides, and covalent adducts in samples from preclinical and clinical kinetic studies

Abstract

A gradient reversed-phase HPLC analysis for the direct measurement of gemfibrozil (GEM) and four oxidative metabolites in plasma and urine of humans and in tissue homogenates of rats was developed. The corresponding acyl glucuronides and the covalently bound protein adducts (in protein precipitates) were determined after liberation from the respective conjugates via alkaline hydrolysis. The limits of detection for the covalent adducts in human plasma are: 10 ng ml⁻¹ (GEM), 20 ng ml⁻¹ (M1), 0.5 ng ml⁻¹ (M2, M4), and 5 ng ml⁻¹ (M3). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy. It has been applied to the analysis of preclinical and clinical studies. Pharmacokinetic profiles of gemfibrozil, its metabolites, and covalent adducts in human plasma and rat tissue homogenates are given.

Introduction

Gemfibrozil, a fibrate lipid-regulating agent, is clinically effective in reducing serum cholesterol and triglyceride levels, decreasing LDL and increasing HDL levels [1]. Apart from new HMG CoA reductase inhibitors (e.g., atorvastatin), gemfibrozil is still a drug of choice in the treatment of hyperlipidaemias involving raised triglyceride levels [2] and has been effective in reducing the incidence of coronary heart disease (by 34%) [1]. Extensive phase-I metabolism yields at least four oxidative metabolites – three hydroxylated products (M1, M2, M4) and one dicarboxylic acid (M3) – and subsequent conjugation with glucuronic acid leads to the five respective acyl glucuronides (Fig. 1) [3], [4], [5], [6]. Renal excretion is the most important elimination pathway for the respective carboxylic acids as well as such glucuronides in man. Accordingly, a total of 60–70% of a gemfibrozil dose are found in urine, while only about 6% are recovered in faeces following peroral administration [4], [5]. As to be expected for lower molecular weight compounds, to which, upon glucuronide formation, almost 200 molecular weight units are added, considerable species differences were detected with respect to the extent of renal and biliary/faecal excretion [7], [8]. In rats, further oxidative metabolites were identified in rat urine [9], including a diol metabolite (both ring methyls hydroxylated) and the product of its further metabolism, the acid–alcohol derivative (ortho ring methyl hydroxylated, meta ring methyl further oxidized to acid), as well as ether and acyl (=ester) glucuronides of the same metabolite.

Acyl glucuronides are reactive intermediates which undergo typical chemical reactions such as hydrolysis, isomerization, and covalent binding to endogenous compounds. Hydrolysis of the ester glucuronides at physiological pH leads to back-formation of the aglycones and may be responsible for a potential increase of aglycone concentrations, also ex vivo/in vitro, i.e., after blood or urine sampling. Since the hydrolysis rate is temperature and pH-dependent, post-sampling degradation can be minimized by immediate cooling and pH-stabilization (pH 3–4) [10], [11], which is essential for the correct determination of aglycone and glucuronide concentrations. Isomerization – i.e., migration of the acyl residue from the C-1 position to the C-2, C-3, and C-4 positions of the glucuronic acid ring – leads to the generation of β-glucuronidase-resistant regioisomers. Like hydrolysis, it is pH-dependent and can hence be avoided. Covalent binding has been demonstrated for many acyl glucuronide-forming drugs in vivo [3], [12], [13], [14], [15], [16], [17], in vitro studies have verified the role of the acyl glucuronides in the formation of such covalent adducts [3], [15], [16], [17], [18], [19], which were proven to be formed via two different mechanisms, i.e., by nucleophilic displacement and condensation of the rearranged acyl glucuronide isomers with lysine residues (e.g. Lys-159), as shown for the acyl glucuronide of benoxaprofen [17], [20].

In addition to pH-dependent isomerization and hydrolysis, tissue homogenates include a further complicating source of instability, which is hydrolysis via lysosomal hydrolytic enzymes that are released upon the homogenization process and exhibit their highest activity at acidic pH, i.e. at the pH level where nonenzymatic hydrolysis and isomerization are minimized.

Analytical methods are described in literature for the quantification of gemfibrozil in plasma (HPLC) [21], [22], for gemfibrozil and its main metabolite M3 in plasma and urine (GC [23], HPLC [24]), and for the respective glucuronides (after hydrolysis). Another method, which was developed for the simultaneous determination (HPLC) of gemfibrozil and M1-M4 in plasma and urine requires the use of two detectors and two integrators as well as different stationary and mobile phases for the different biophases (plasma, urine) [4]. Moreover, it was only validated for fairly high plasma concentrations, so that the determination of the usually low concentrations of M2 and M4 was not possible. Furthermore, in the current studies, the respective procedure was expanded to include compounds released from the respective acyl glucuronides and covalent protein adducts.

Thus, aim of the present investigations was (a) to establish a direct gradient HPLC method for the simultaneous determination of gemfibrozil and M1–M4 in concentration ranges relevant for pharmacokinetic studies, and to validate the assay for the measurement of gemfibrozil, its major phase-I and phase-II metabolites, and the respective covalent adducts in different biophases (human plasma and urine, various rat tissues) as well as (b) to apply the methods to the elucidation of the pharmacokinetics of gemfibrozil, all its metabolites, and adducts in humans and rats.