N-Glucuronidation of Drugs and Other Xenobiotics

UDP-glucuronosyltransferases (UGTs) are a family of metabolic enzymes responsible for the detoxification of a wide range of endo- and xenobiotics, including drugs. UGT-mediated metabolism is a major determinant of the pharmacokinetic behavior of many drugs in the human body, contributing to parameters such as bioavailability and elimination. UGTs catalyze the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to the aglycone substrates, producing water-soluble glucuronide conjugates that are mostly devoid of pharmacological activity. Some of the 19 human UGTs have the ability to conjugate different nitrogen-containing compounds, thus forming N-glucuronides. N-Glucuronidation exhibits marked differences across species. As an example, the ability to form quaternary ammonium glucuronides from tertiary amines is a reaction largely, but not completely, restricted to humans. Among the human UGTs, UGT1A4 has been considered the enzyme specializing in N-glucuronidation. The goal of this study was to characterize species differences related to N-glucuronidation and to elucidate whether human UGT enzymes other than the previously reported UGT1A4 catalyze this reaction. The nitrogen-containing substrates investigated were firstly a set of 4-arylalkyl-1H-imidazoles, including the sedative drug dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole], and secondly nicotine, the addictive agent in tobacco products, along with its major metabolite cotinine. The study was performed using different in vitro systems, such as human and animal liver microsomes and recombinant human UGT enzymes produced in baculovirus-infected insect cells. The 4-arylalkyl-1H-imidazole substrates were incubated with a radiolabeled cofactor, 14C-UDPGA, and the glucuronide products were analyzed by liquid chromatography using ultraviolet detection combined with a flow scintillation analyzer. Nicotine and cotinine glucuronides were quantified using liquid chromatography mass spectrometry. Analyses of liver microsome incubates indicated that N-glucuronidation of medetomidine was efficient in humans, while the glucuronidation rates measured in rat, mouse, guinea-pig, rabbit, dog, mini-pig, and monkey liver microsomes were rather low. Studies with recombinant human UGTs revealed that the orphan enzyme UGT2B10, which has previously demonstrated no or only very limited activity when screened against a large variety of substrates, plays an important role in the N-glucuronidation of the compounds investigated. More specifically, we discovered that UGT2B10 is the enzyme mainly responsible for nicotine N-glucuronidation in the human liver, and, furthermore, this enzyme is a major contributor to the N-glucuronidation of medetomidine. Nuclear magnetic resonance (NMR) analyses revealed that the N-glucuronidation of medetomidine by human liver microsomes was highly regioselective, and that N3 was the preferred site of glucuronidation. Moreover, this chiral drug was N-glucuronidated stereoselectively. Regio- and stereospecific N-glucuronidation of medetomidine in human liver microsomes was explained by complex kinetics involving two enzymes, UGT1A4 and UGT2B10. UGT2B10 was found to be a high-affinity (low Km) enzyme towards medetomidine, while the affinity of UGT1A4 was considerably lower. Levomedetomidine [( )-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole], in particular, turned out be a high-affinity, specific substrate of UGT2B10. The results emphasize the species differences of N-glucuronidation and the importance of in vitro studies utilizing human-derived material, along with animal studies, at the early stages of drug development. Furthermore, this study highlights the contribution of UGT2B10, along with UGT1A4, to the N-glucuronidation of drugs and other xenobiotics in the human liver.Lääkekehityksen tavoitteena on löytää tehokkaita ja turvallisia uusia lääkeaineita. Lääkeaineiden aineenvaihdunnan eli metabolian selvittäminen on tärkeä osa tätä prosessia. Metabolia on elimistössä, lähinnä maksassa, tapahtuvaa lääkeaineiden biokemiallista muuntumista ns. metaboloivien entsyymien katalysoimana. Metabolia toimii puolustusmekanismina elimistöön ulkopuolelta tulevia kemikaaleja vastaan muuttaen näitä vierasaineita, kuten lääkeaineita, yleensä vesiliukoisempaan ja harmittomampaan muotoon. Metabolian seurauksena syntyy metaboliitteja, jotka erittyvät elimistöstä virtsaan tai ulosteisiin päättäen lääkeaineen farmakologisen vaikutuksen. Voimakas metabolia maksassa saattaa olla este lääkkeen teholle, jos lääkkeen pitoisuudet metabolian seurauksena jäävät lääkkeen vaikutuskohdassa liian pieniksi. Metabolia on usein myös lääkkeiden yhteisvaikutusten taustalla, sillä monet lääkeaineet metaboloituvat samojen entsyymien välityksellä. Uridiinidifosfoglukuronosyylitransferaasit (UGT:t) ovat osa elimistön metaboliakoneistoa. Nämä entsyymit katalysoivat vesiliukoisen sokerin, uridiinidifosfoglukuronihapon (UDPGA), liittämistä lääkeaineisiin. Reaktion seurauksena syntyvää metaboliittia kutsutaan glukuronidiksi. Monet erityyppiset lääkeaineet erittyvät elimistöstä pääasiallisesti glukuronideina. Tässä työssä tutkittiin N-glukuronidaatiota eli UDPGA:n konjugoitumista lääkeaineen aminoryhmään käyttämällä malliaineina joukkoa 4-aryylialkyyli-1H-imidatsoleita kuten deksmedetomidiinia, jota käytetään tehohoidossa potilaiden kivunlievitykseen ja nukutukseen. Lisäksi tutkittiin nikotiinia, joka on tupakan riippuvuutta aiheuttava kemikaali, sekä tämän päämetaboliittia kotiniinia. Tutkimus toteutettiin käyttäen erilaisia in vitro tekniikoita kuten ihmisen ja eri eläinlajien maksakudoksesta eristettyjä mikrosomeja sekä geenitekniikalla hyönteissoluissa tuotettuja ihmisen UGT-entsyymeitä. Näiden avulla pyrittiin selvittämään lajien välisiä eroja N-glukuronidaatiossa sekä sitä, mitkä ihmisen UGT-entsyymit metaboloivat malliaineita. Tutkimuksessa havaittiin, että malliaineiden metabolia N-glukuronideiksi oli ihmisen maksassa tehokkaampaa kuin millään tutkituista eläinlajeista. Tutkituista ihmisen 16 UGT-entsyymistä UGT1A4 ja UGT2B10 metaboloivat malliaineita. Etenkin UGT2B10 osoittautui tärkeäksi sekä deksmedetomidiinin että nikotiinin metaboliassa ihmisen maksassa. Tutkimustulokset auttavat ymmärtämään N-glukuronidaation välityksellä metaboloituvien lääkeaineiden metaboliassa havaittuja suuria lajieroja. Ihmisen ja eläinten välisten metaboliaerojen tunteminen on tärkeää mm. lääkeaineiden turvallisuustutkimusten tulosten tulkitsemisessa. Tutkimuksessa kehitetyt in vitro menetelmät soveltuvat myös muiden lääkeaineiden metaboliaominaisuuksien seulontaan lääkekehityksen alkuvaiheessa

Expression of UDP-glucuronosyltransferase 1A4 in human placenta at term

The placenta contains a large variety of metabolizing enzymes, among them UDP-glucuronosyltransferase (UGT). Several UGT2B isozymes have so far been detected in human placenta, but little is known on placental expression of UGT1A isozymes. The antiepileptic drug lamotrigine (LTG) is a UGT1A4-substrate, and its serum concentration falls by over 50% during pregnancy, leading to impaired seizure control. The placenta may be involved in this. Microsomes from term placentas of 4 LTG-users and 10 healthy control subjects were prepared. Western blot analysis detected UGT1A proteins in all placentas. The presence of UGT1A4 in placenta from LTG users was confirmed with UGT1A4 commercial standard and a specific UGT1A4 primary antibody. Since LTG is primarily metabolized by UGT1A4 and this isozyme is shown to be present in placenta at term, it may be hypothesized that the placenta is involved in the fall of LTG serum concentrations during pregnancy

Current trends in drug metabolism and pharmacokinetics.

Pharmacokinetics (PK) is the study of the absorption, distribution, metabolism, and excretion (ADME) processes of a drug. Understanding PK properties is essential for drug development and precision medication. In this review we provided an overview of recent research on PK with focus on the following aspects: (1) an update on drug-metabolizing enzymes and transporters in the determination of PK, as well as advances in xenobiotic receptors and noncoding RNAs (ncRNAs) in the modulation of PK, providing new understanding of the transcriptional and posttranscriptional regulatory mechanisms that result in inter-individual variations in pharmacotherapy; (2) current status and trends in assessing drug-drug interactions, especially interactions between drugs and herbs, between drugs and therapeutic biologics, and microbiota-mediated interactions; (3) advances in understanding the effects of diseases on PK, particularly changes in metabolizing enzymes and transporters with disease progression; (4) trends in mathematical modeling including physiologically-based PK modeling and novel animal models such as CRISPR/Cas9-based animal models for DMPK studies; (5) emerging non-classical xenobiotic metabolic pathways and the involvement of novel metabolic enzymes, especially non-P450s. Existing challenges and perspectives on future directions are discussed, and may stimulate the development of new research models, technologies, and strategies towards the development of better drugs and improved clinical practice

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density

Introduction Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Methods Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. Results SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Conclusions Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer

Naishiihtäjien representoinnin muutokset Urheilulehden teksteissä vuosina 1905-2010

The purpose of my study is to examine how female skiers are represented in the texts of a Finnish sports magazine, Urheilulehti, and how these representations have changed during the examination period from 1905 to 2010. Relying on Judith Butler’s ideology of gender performativity, I analyse the representations of female skiers from the perspective of promoting the traditional female norm and simultaneously from the perspective of breaking and reforming it. The methodological framework of my study consists of Fairclough’s three- dimensional framework, which is based on the tradition of critical discourse analysis (CDA). In compliance with this framework, I began the analysis from the textual level and proceeded through the discursive practice level to explaining different interpretations at the social practice level. The analysis produced a classification of four ‘upper-level discourses’ and eight ‘main discourses’ for structuring female skiers’ representations. The upperlevel discourses were named skier discourse, female skier discourse, individual discourse and female discourse. The main discourses are based on pairs of opposing discourses within each upper-level discourse: successful skier – failing skier, respected female skier – unaccepted female skier, worthy individual – unworthy individual, and traditional woman – boundary-breaking woman. Several changes were discovered in the representations of female skiers. The discourse of successful skier, which became the dominant discourse in the examined texts, increased its share towards the end of the research period. During the last years, it became increasingly common to represent female skiers even as sports heroes. Descriptions of female skiers’ personal lives began to appear in the texts in 1970, and the role of these descriptions grew towards the end. Texts highlighting traditional femininity became less frequent, whereas descriptions of deviation from the traditional female norm increased. Overlapping of the discourses became typical of the representations. In the last years of the data, female skiers were primarily presented as multidimensional ‘subjects’ or agents. Contrary to earlier research findings, the texts in the final years of the research period included no trivialisation of female skiers, such as underestimation, description of appearances or focus on irrelevant matters