The human UDP-glucuronosyltransferase UGT1A3 is highly selective towards N2 in the tetrazole ring of losartan, candesartan, and zolarsartan
Introduction
Losartan, candesartan, and zolarsartan (GR117289, also called zolasartan) inhibit the effect of angiotensin II by acting as AT1 receptor antagonists. Losartan and candesartan are in clinical use for the treatment of hypertension. In addition, it was recently reported that losartan might be effective in treating Marfan syndrome and perhaps other illnesses [1], [2], [3], [4]. Zolarsartan is also an active compound both in vitro [5] and in vivo [6]. Losartan itself is not as active as its carboxylic acid metabolite, which is also longer acting. Candesartan is administered as a prodrug, candesartan cilexetil, which has better bioavailability than candesartan. Candesartan cilexetil is hydrolyzed to the active candesartan during the absorption process. The molecular structures of losartan, candesartan, and zolarsartan include a tetrazole moiety, while candesartan and zolarsartan also have a carboxylic acid group (Fig. 1). Losartan is metabolized by three main routes: oxidation of the alcohol to carboxylic acid, hydroxylation, and glucuronidation [7]. Candesartan is mostly excreted unchanged [8], but glucuronide conjugates have been found from rats and dogs [9], [10]. Rats and dogs also produce zolarsartan glucuronides [11].
Glucuronidation is an important phase II metabolic reaction. UDP-glucuronosyltransferases (UGTs) are membrane bound enzymes that catalyze this conjugation reaction [12], [13], [14], [15], [16]. Glucuronidation is an SN2 reaction where the nucleophilic substrate attacks the uridine-5′-diphospho-α-d-glucuronic acid (UDPGA). The most common glucuronidation products are O- and N-glucuronides formed from hydroxyl, carboxyl, or amino functional groups, but there are also reports of S- and C-glucuronides [12], [16], [17].
The human UGTs are divided into three subfamilies, UGT1A, UGT2A, and UGT2B, based on their amino acid sequences and gene structures [14], [18]. UGTs catalyze the glucuronidation of a wide range of endogenous and exogenous compounds, including steroid hormones, dietary constituents, and drugs. The substrate specificity of most UGTs is wide and often partly overlapping. Most human UGTs catalyze the glucuronidation of hydroxyl or carboxyl groups in the form of ether or acyl O-glucuronides, whereas N-glucuronides, formed from various amines, are produced by much fewer UGTs [12], [16], [19].
Losartan can be glucuronidated at three different positions so that one O-glucuronide and two different tetrazole-N-glucuronides may be formed [20] (Fig. 1). The O-glucuronide and one of the N-glucuronides, tetrazole-N2-glucuronide, were found to be formed during biotransformation and tetrazole-N1-glucuronide has been synthesized both chemically [20] and enzymatically [21]. The glucuronidation of losartan has been previously studied using liver slices [7], [20] and liver microsomes [22] from various species, as well as rat intestine [23]. Monkeys and rats produced both O-glucuronide and tetrazole-N2-glucuronide whereas humans and dogs produced only the tetrazole-N2-glucuronide of losartan. The metabolism of candesartan cilexetil has been studied in rats and dogs, yielding both the O-glucuronide (acyl glucuronide) and the tetrazole-N2-glucuronide of candesartan [9], [10] (Fig. 1). Tetrazole-N1-glucuronides of candesartan have not thus far been found. Zolarsartan can be glucuronidated to O-glucuronide in rats, while dogs, in addition to acyl glucuronide, also produce tetrazole-N2-glucuronide [11] (Fig. 1). Zolarsartan N1-glucuronide has been synthesized enzymatically using rat liver microsomes as a catalyst [21].
The aim of this study was to examine the glucuronidation of the aglycones losartan, candesartan, and zolarsartan by the human UGTs of subfamilies 1A and 2B. We have selected the three substrates on the basis of similarity of structure, i.e. all contain a tetrazole, and in addition both O- and N-glucuronides can be formed without a phase I metabolism reaction. Among other things, we wanted to find out if the tetrazole group, common to all three substrates, was glucuronidated by the same UGT isoform(s). In addition, liver microsomes from human, bovine, moose, pig, rabbit, and rat were studied to explore species differences in glucuronidation of sartans.
Section snippets
Reagents
Losartan potassium (98% purity), candesartan (purity not reported), and zolarsartan (purity not reported) were obtained from Merck (Rahway, NJ), AstraZeneca (Mölndal, Sweden), and GlaxoSmithKline (Hertfordshire, UK), respectively. Glucuronide regioisomers of losartan, candesartan, and zolarsartan were synthesized previously in our laboratory and their structures were characterized by nuclear magnetic resonance spectroscopy (NMR) [21]. Saccharic acid 1,4-lactone and UDPGA (trisodium salt, 99.8%
Results
We have recently biosynthesized and characterized by NMR the different glucuronides of the three sartans that are included in the current study [21]. The glucuronidation assays used here were carried out using a validated HPLC method. The results of the repeatability experiments are presented in Table 2, and they demonstrated that this method gave reliable analyses of the samples. In particular, the losartan N2-glucuronide standard curve that was used for quantitation in the kinetic assays, was
Discussion
The glucuronidation of losartan, candesartan, and zolarsartan by different animal liver microsomes and a large set of recombinant human UGTs was studied. An interesting aspect of these sartans is the presence of several potential sites for both N- and O-glucuronidations within each aglycone (Fig. 1). We have recently characterized the structures of the N- and O-glucuronides that are produced from these sartans by NMR [21], providing a tool to study the regioselectivity of individual UGTs in
Acknowledgments
We thank Merck, AstraZeneca, and GlaxoSmithKline for generously providing losartan, candesartan, and zolarsartan (GR117289), respectively. Katriina Itäaho, Sirkku Kallonen, Mika Kurkela, Johanna Mosorin, and Sanna Sistonen are acknowledged for helpful discussions, help with GraphPad Prism and LC–MS analyses, as well as for determination of expression levels of UGTs and other technical assistance. This study was financially supported by the Graduate School in Pharmacy (AA) and by the Academy of
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