Deferiprone Glucuronidation by Human Tissues and Recombinant UDP Glucuronosyltransferase 1A6: An in Vitro Investigation of Genetic and Splice Variants

Abstract

ABSTRACT:Tissue iron overload constitutes a major health problem for people who require regular blood transfusions, such as those with ␤-thalassemia major. Deferiprone is a hydroxypyridinone iron chelator used therapeutically to remove this excess iron and prevent tissue damage. Deferiprone is metabolized by UDP-glucuronosyltransferases (UGTs) into deferiprone 3-O-glucuronide (DG), but a systematic evaluation of the contribution of individual human UGTs and the impact of genetic variations of UGTs have not been conducted. Sixteen human UGT1A and UGT2B were studied for deferiprone glucuronidation, and their clearances were compared in human tissue samples. DG was measured by liquid chromatography coupled with mass spectrometry. DG was primarily produced in vitro by UGT1A6, and a second glucuronide metabolite was discovered. UGT1A6, as well as liver and kidney human microsomes, had similar kinetic profiles and clearance (Cl int ‫؍‬ 1.4-3.0 l/min/mg), but clearance by intestinal microsomes was much lower (0.04 l/min/mg). Binding of deferiprone to microsomal preparations was not significant. Genetic variants of UGT1A6 had K m values similar to the reference protein (UGT1A6*1), but their V max values were reduced by 25 to 70%. The UGT1A6 splice variant isoform 2, detected in the liver and kidney, had no transferase activity for deferiprone. When UGT1A6_i2 was coexpressed with the classic UGT1A6_i1 isoform, velocity was reduced for deferiprone but remained similar for 4-nitrophenol or serotonin glucuronidation. In conclusion, deferiprone glucuronidation seems to depend almost totally on UGT1A6, especially in the liver. Genetic variations and differences in the expression of splice variants represent a potential source of variation in deferiprone metabolism.

In Vitro Glucuronidation of Fenofibric Acid by Human UDP-Glucuronosyltransferases and Liver Microsomes

“…In contrast to a previous study that showed recombinant UGT1A10 homogenates have greater activity compared with UGT1A10 microsomes for a variety of substrates, including tamoxifen Sun et al, 2007), we observed that microsomes are more active for fenofibrate than homogenates, suggesting that such differences could be substrate-specific. A higher activity with microsomes compared with Supersomes was also observed, as noted in other studies (Benoit-Biancamano et al, 2009). Thus, a primary role of UGT2B7 followed by UGT1A9 and possibly UGT1A3 in the hepatic clearance of FA is predicted and further supported by correlative studies using liver microsomes and UGT probe substrates.…”

Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

“…Tacrolimus (200 mM, 60 minutes) was obtained from Cell Signaling Technologies (Danvers, MA). Glucuronides were measured by mass spectrometry-based methods (Lepine et al, 2004;Benoit-Biancamano et al, 2009).…”

A Novel Method for the Immunoquantification of UDP-Glucuronosyltransferases in Human Tissue

“…A proportion of the variability of UGT1A1 expression in the population is due to the common variable number tandem repeat polymorphism in the UGT1A1 gene promoter associated with Gilbert's syndrome (Bosma et al, 1995;Monaghan et al, 1996) [or other mutations in different populations, e.g., Koiwai et al (1995))]; however, measurements of mRNA levels show very wide interindividual variation for all UGTs [e.g., Izukawa et al (2009))], suggesting that other environmental, genetic, and/or epigenetic factors significantly affect expression levels. UGT1A6 is responsible for the glucuronidation of many xenobiotics, and several drugs are known to be substrates for this enzyme, including paracetamol (Court et al, 2001), valproate , and deferiprone (Benoit-Biancamano et al, 2009). UGT1A6 also metabolizes several important endogenous substrates such as serotonin (Krishnaswamy et al, 2004).…”