Browsing by Author "Alonen, Anna"

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  • Alonen, Anna (Helsingin yliopisto, 2009)
    Drug metabolism reactions are divided into two phases. Glucuronidation is an important phase II metabolism reaction that is catalyzed by UDP-glucuronosyltransferases (UGTs). Due to glucuronic acid conjugation, bioactive compounds become inactive, and lipophilic substances become more hydrophilic, which enhances their excretion to urine and bile. Aglycones can be endogenous or exogenous compounds, such as hormones and drugs. Synthetic glucuronide metabolites are needed as reference substances in drug research. Glucuronide conjugates have been synthesized by both chemical and enzymatic methods. The aim of this work was to study glucuronidation of aglycones that have multiple glucuronidation sites. Glucuronide isomers of dobutamine, losartan, candesartan, and zolarsartan were synthesized by enzyme-assisted methods whereas clenbuterol glucuronides were synthesized chemically using the Koenigs-Knorr reaction. The structures and the glucuronidation sites of the synthesized compounds were determined by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. From dobutamine, the glucuronide synthesis produced three O-glucuronide regioisomers. Both N- and O-glucuronides, including two acyl glucuronides, of losartan, candesartan, and zolarsartan were obtained. The Koenigs-Knorr reaction produced O-glucuronide stereoisomers of clenbuterol. The yields varied between 0.7 and 34.6% (0.5 6.8 mg). It is important to identify active UGT isoforms in order to understand drug-drug interactions, to clarify catalytic mechanisms, to assess the structure-function relationships, and to find isoform-selective probes. In this study, the glucuronidation activity of recombinant human UGTs was assessed using the synthesized glucuronides as reference compounds. In addition, species differences in glucuronidation were studied using liver microsomes from various animals. The formation of glucuronide isomers of dobutamine, losartan, candesartan, and zolarsartan brought a new perspective to evaluation of the structure-function relationships of UGTs and the results provided new data on their substrate specificities. UGT1A10 showed versatile substrate specificity whereas UGT1A3 was highly selective towards N2 in the tetrazole ring. The glucuronidation activity of liver microsomes was different between species. Compared to animal liver microsomes, human liver microsomes glucuronidated the studied aglycones inefficiently. There were no distinct correlations between the glucuronidation properties of human liver microsomes and recombinant hepatic UGTs, so it is difficult to predict the glucuronidation in human liver microsomes from the data obtained with recombinant UGTs.