Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS


SGLC/MS Streszczenie raportu

Project ID: G6RD-CT-2001-00513
Źródło dofinansowania: FP5-GROWTH
Kraj: Finland

Enzymatic synthesis methods for phase II metabolites of anabolic androgenic steroids

Conjugation with glucuronic acid is the major conjugation reaction in all mammals. Various functional groups have the potential of reacting with glucuronic acid to form O-, S-, N-, and C-glucuronides, which means that a wide variety of compounds are metabolized via the glucuronidation pathway. Glucuronidation is a bimolecular nucleophilic substitution (SN2) reaction, which is catalyzed by uridine diphosphoglucuronosyltransferases (UGTs; Enzyme Classification E.C. and uses uridine-5’-diphosphoglucuronic acid (UDPGA) as the co-substrate. The reaction leads to the attachment of the polar sugar moiety to the substrate, which in the case of anabolic androgenic steroids (AAS) is phase-I transformed metabolite of the parent compound, with the immediate inversion of the configuration to yield a beta-glycosidic bond. As a result, these metabolic reactions in most cases terminate the activity of xenobiotics and endobiotics, including AAS. The main site of glucuronidation is the liver, although extra-hepatic glucuronidation has been observed in kidney, intestines, lung, and prostate.

UGTs are a family of enzymes bound in the membrane of the endoplasmic reticulum, which catalyze the glucuronidation of various endogenous and exogenous compounds, including steroids. At least 16 different UGTs, ranging from 526 to 533 amino acids in size, are encoded by the human genome. The highly homologous carboxyl terminal is suggested to contain the domain critical for catalysis and for binding of UDPGA, whereas the amino terminal is responsible for the substrate specificity. The expressed UGT proteins have been categorized into two families (UGT1 and UGT2) on the basis of the protein sequence similarity, which is higher than 38% within a single family. According to the sequence homology, the enzyme families are further divided into subfamilies. The most important enzymes involved in steroid glucuronidation are members of subfamilies UGT1A and UGT2B.

Scientific result of the enzyme-assisted synthesis offers an elegant and alternative method for the production of anabolic steroid glucuronides parallel to the conventional chemical synthesis pathway. The advantage of the enzyme-mediated synthesis route is the stereo-selectivity encountered in nature for biochemical catalyst, i.e. UGTs. Methods using rat liver preparations as a source of conjugating enzymes have been described for a structurally diverse group of substrates, e.g. p-nitrophenol, a series of nitrogen-containing antidepressants, nitrocatechols, as well as for endogenous steroids such as androsterone, androstanediol, dihydrotestosterone, epitestosterone, and testosterone.

Within this project a representative group of anabolic steroids, either the parent steroids or their phase-I transformed metabolites were selected for the enzyme-assisted synthesis, having 3-alpha, 17-alpha, or 17-beta-oriented hydroxyl group(s) in various combinations available as potential glucuronidation sites. Arochlor-induced rat liver microsomal UGTs were used in the synthesis of AAS glucuronides and despite the differences between the conjugation activities, all the tested compounds were capable of glucuronidation in these experiments. Relative glucuronidation rates were higher with rat than human liver microsomal preparations, owing most probably to the enzyme-induction of the rat preparations. In general comparison the activities of liver preparations were significantly higher than those of the tested recombinant UGT isoenzymes, which may be at least partly explained by the better tolerance of tissue preparations to organic solvents in the synthesis mixture.

Glucuronide-conjugated metabolites are needed as reference compounds in several fields of analytical research, such as in forensic, metabolic, and pharmaceutical laboratories. In many cases, especially with the new chemical entities, the lack of suitable commercial products represents a major problem in the research progress. Enzyme-assisted synthesis offers a practical pathway for the rapid production of small amounts of AAS glucuronides, such as needed in the build-up of an analytical method. Because the composition of the reaction mixture is relatively simple, and the differences in the optimal conditions for the various substrates are relatively minor, the addition of a new AAS substrate to the test compound set should be straightforward. And furthermore, the substrates are not limited only to AAS, but the methods can be applied to the first steps of conjugation studies also for the substrates with different structures. In future, an appropriate selection of UGT isoenzymes might be used as in vitro model to predict the glucuronidation reactions of a particular xenobiotic in the human body, but for that, the metabolic patterns should be examined in tissues to ensure the equivalence of the isoenzymes.

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University of Helsinki
Viikinkaari 5E
00014 Helsinki
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