Short communicationNMR spectroscopic studies of the transacylation reactivity of ibuprofen 1-β-O-acyl glucuronide
Introduction
There is a continuing interest in the transacylation reactivity of drugs, which form ester glucuronides that might be associated with toxicity. The development of quantitative structure-reactivity relationships (QSRRs) for this group of compounds could be of potential benefit when designing new drugs as with an appropriate model it might be possible to eliminate the potential for reactivity from new compounds. As part of research into the QSRRs of these metabolites, the degradation rates of a series of model glucuronides (based on substituted benzoic acids) have been determined and the results used to build several promising QSRRs based on both experimental and calculated properties, e.g. 13C chemical shifts, partial atomic charges on the ester carbonyl carbons and the Hammett substituent parameter, σp [1], [2].
Drugs with a carboxylic acid functional group, such as the non-steroidal anti-inflammatory drug (NSAID) ibuprofen, can readily form 1-β-O-acyl glucuronides in vivo, and this represents a major route for the metabolism and thence elimination of such compounds [3], [4], [5]. Depending on the nature of the aglycone, 1-β-O-acyl glucuronides can be unstable in aqueous solution under acidic, neutral and alkaline pH conditions and exhibit a range of reactivities [4]. Under physiological conditions, at pH 7.4, the carbonyl carbon of the acyl ester is susceptible to nucleophilic attack. The 1-β-O-acyl glucuronide can hydrolyse, liberating the aglycone, but importantly the molecule can undergo an intra-molecular rearrangement, whereby the hydroxyl group at C2 of the glucuronic acid moiety can attack the acyl carbonyl carbon to form a cyclic ortho-ester intermediate. This can then ring open to form the 2-O-acyl ester. The acyl migration reaction can then proceed in a similar fashion to produce 3-O- and 4-O- position esters. The glucuronide ring of 2-, 3- and 4-O-acyl positional isomers can open and recyclise via the aldehyde form, enabling mutarotation to occur yielding the α- and β-anomers of the isomers. Experiments have also shown the formation of 1-α-O-acyl glucuronides in vitro and these are presumably formed from a α-2-O-acyl glucuronide (themselves formed after transacylation from the 1-β-O-acyl anomers followed by mutarotation of the β-2-O-acyl isomers) [6]. The analogous reaction of formation of 1-β-O-acyl from the β-2-O-acyl isomers (again via the cyclic ortho-ester intermediate with 1,2-trans-(eq,eq) fused rings) cannot be ruled out in light of this new evidence, and was first proposed on the basis of HPLC/UV analysis of diflunisal 1-β-O-acyl glucuronides and enzyme hydrolysis experiments [7]. The full reaction scheme has been shown several times previously [1], [2].
The resulting transacylation equilibrium mixture contains six positional isomers and anomers, plus residual amounts of the 1-β-O-acyl/1-α-O-acyl isomers formed by back-reaction. In addition, all of these isomers are, irreversibly, undergoing hydrolysis. The resulting glucuronides (particularly the isomers in the 3- and 4-position) may act as haptens and stimulate immune responses/allergic reactions in vivo as they can react to form covalently bound adducts with cellular macromolecules, via imine formation/Amadori rearrangement or nucleophilic displacement [3], [4], [5]. Circumstantial evidence suggests that drugs that undergo these rapid acyl migrations appear to be related to the incidence of hypersensitivity in clinical use, and these reaction products have also been linked to hepatotoxicity [5], [8]. Measurable covalent binding to human serum albumin in vitro and to mouse hepatic proteins by the 1-β-O-acyl glucuronides of a number of drugs has been shown [8], [9], [10], [11].
For the simplest situation in this reaction scheme, the chemical degradation of the 1-β-O-acyl glucuronide, there are two parallel reactions, namely acyl migration which is a unimolecular first order process, and hydrolysis which is a second order reaction but because [H2O] ≫ [1-β-O-acyl glucuronide] this is also essentially a first order process. The rate at which the 1-β-O-acyl component is depleted – the degradation rate (kd) – is therefore the sum of these two reaction rates. This rate can be easily measured by following the disappearance of the 1H NMR resonance arising from the anomeric proton of the 1-β-O-acyl isomer as the reaction progresses in an NMR tube at pH 7.4.
HPLC could also be used to monitor the reaction using UV detection but that this does not provide the molecular identification that the somewhat slower NMR method can yield. In addition, for drug glucuronides of widely varying log P values, different HPLC conditions have to be used and this can make comparisons between compounds difficult and thus can minimise the usefulness of the results for structure-reactivity relationship development.
The metabolism of ibuprofen has been studied extensively and the major metabolic products have been identified as the oxidation products hydroxyl-ibuprofen and carboxyl-ibuprofen and the acyl glucuronides of these as well as of ibuprofen itself [12], [13]. The drug is administered as a racemate but there is considerable epimerisation in vivo and the ratio of S-ibuprofen to R-ibuprofen glucuronide in plasma is about 7.1 [14]. It has been shown in vitro that ibuprofen glucuronide is labile and can form covalent adducts with proteins, and in the case of elderly patients receiving long term administration of ibuprofen, covalent binding to plasma proteins has been shown in vivo [15].
Here we have measured the degradation rate of ibuprofen glucuronide in vitro and have compared the value obtained with those from other NSAID glucuronides.
Section snippets
Chemicals
All chemicals were obtained from Sigma–Aldrich Ltd. (Dorset, UK) and used as received. C18 mega Bond-Elut™ SPEC columns (12 ml reservoir volume/2 g sorbent mass) were obtained from Varian Ltd., (Walton-on-Thames, UK).
A sample of urine was collected 7 h following administration of a single oral dose of 200 mg of ibuprofen to a healthy male subject. The sample (∼300 ml) was then acidified to pH 5.6 using 0.1 M hydrochloric acid and stored at −40 °C until required. A C18 Bond-Elut® column (Varian) was
Results
The 1-β-O-acyl glucuronide of ibuprofen was isolated using solid-phase extraction chromatography (SPEC) and characterised by NMR spectroscopic analysis [1]. After administration of racemic ibuprofen it has been reported that the drug excreted in human urine is largely in the (S)-form, due to chiral inversion in vivo. If both R- and S-ibuprofen glucuronides had been present in the sample (as a diastereoisomeric mixture) [14] a pair of doublets would have been observed for the anomeric proton
Discussion
The isolation of glucuronides using SPEC extraction techniques proved to be relatively easy for ibuprofen and, once a suitable solvent elution system had been developed, provided a rapid route to providing the target compound in quantities and purity suitable for analysis.
The synthesis, kinetic studies and QSRR results for a series of benzoic acid glucuronides compounds have also been described recently [1], [2] and it would be interesting to extend this approach to arylpropionic acid
Acknowledgements
The BBSRC and AstraZeneca are thanked for financial support (SJV). We would also like to thank Dr Olivia Corcoran (University of London) and Dr Claire Gavaghan (Imperial College) for useful discussions regarding the analysis of O-acyl glucuronides.
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