Biosynthesis of drug glucuronides for use as authentic standards.
BACKGROUND
Glucuronidation by the uridine diphosphate glucuronosyltransferases (UGTs) plays a pivotal role in the clearance mechanism of both xenobiotics and endobiotics. The detection of glucuronides at low micromolar concentrations is required to accurately model in vitro enzyme kinetics and in vivo pharmacokinetics. However, relatively few glucuronides are currently available as standards for developing liquid chromatography and mass spectroscopy (LC/MS) bioanalytical methods.


METHODS
The glucuronidation capacity of hepatic microsomes prepared from rat (RLM), dog (DLM), monkey (MLM), and human (HLM) was examined for five xenobiotic substrates. In each case, glucuronide standards were produced using the enzyme source most efficient for the production of that specific glucuronide.


RESULTS
Dog hepatic microsomes were used to produce glucuronides for anthraflavic acid (yield: 14 mg), buprenorphine (yield: 14 mg), and octylgallate (total yield: 13 mg), whereas propofol glucuronide (yield: 20 mg), and ethinylestradiol glucuronide (yield: 8 mg) were prepared using HLM. All glucuronides were characterized by LC/MS/MS and nuclear magnetic resonance (NMR) spectroscopy.


CONCLUSIONS
The multimilligram quantities of glucuronide standards produced by this method have many applications throughout drug discovery and toxicology. In addition to allowing the quantification of glucuronide formation from in vitro and in vivo studies, the authentic standards produced could also be used to assess potential pharmacological or toxicological effects of metabolites.
Soars
Matthew G
MG
Department of Drug Disposition, Lilly Research Laboratories, Drop Code 0714, Eli Lilly and Company, Indianapolis, IN 46285, USA. soars_mattew_g@lilly.com
Mattiuz
Edward L
EL
Jackson
David A
DA
Kulanthaivel
Palaniappan
P
Ehlhardt
William J
WJ
Wrighton
Steven A
SA
D000818:Animals; D000880:Anthraquinones / Q000378:metabolism; D002047:Buprenorphine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004997:Ethinyl Estradiol / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D020719:Glucuronides / Q000032:analysis / Q000096:biosynthesis*; D014453:Glucuronosyltransferase / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000378:metabolism*; D015742:Propofol / Q000378:metabolism; D051381:Rats; D012015:Reference Standards; D015262:Xenobiotics / Q000378:metabolism*
D000880:Anthraquinones; D020719:Glucuronides; D015262:Xenobiotics; C016627:octyl gallate; D002047:Buprenorphine; D004997:Ethinyl Estradiol; D005707:Gallic Acid; D014453:Glucuronosyltransferase; C034889:2,6-dihydroxyanthraquinone; D015742:Propofol