Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Abstract

The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3'-azido-3'-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mM) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent Km and Vmax revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest.

Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes.

“…AZT has been reported to be a substrate of the UGT2 isoform (31), although this finding requires further confirmation (16). The rate of formation of GAZT was more than twofold greater in human than in rat microsomes, in agreement with previous reports (27). AZT glucuronidating activity in Gunn rats was comparable to normal rats, and that in CN-I patients was comparable to normal humans.…”

“…AZT glucuronidation assay. This assay was modified from that described previously (27,28). Microsomes were preincubated with OLPC (0.8% wt/wt, OLPC/microsomal protein) at 4 Њ C for 20 min.…”

The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): Initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation

“…The majority of these studies involve the in vitro production of glucuronides in which substrates are incubated with UDPGA and microsomal fractions containing UDPGTs and the products are then analyzed using liquid chromatography [10][11][12][13]. Solubilized microsomal UDPGT has also been immobilized through covalent immobilization on Sepharose beads [14,15] and by entrapment into algenate beads in the presence of polyethyleneimine [16]. The resulting immobilized enzyme reactors (UDPGT-IMERs) retained enzymatic activity, but could not be used in-line in HPLC systems.…”

Human and Rat Liver UDP-Glucuronosyltransferases Are Targets of Ketoprofen Acylglucuronide

“…Ketoprofen glucuronide was prepared from PB-treated rat liver microsomes immobilized on alginate beads, as previously described (Haumont et al, 1991). Semipreparative HPLC was used to separate large amounts of biosynthesized glucuronide from UDP-GlcUA and ketoprofen.…”