Abstract
Raloxifene, a selective estrogen receptor modulator used for the treatment of osteoporosis, undergoes extensive conjugation to the 6-β- and 4′-β-glucuronides in vivo. This paper investigated raloxifene glucuronidation by human liver and intestinal microsomes and identified the responsible UDP-glucuronosyltransferases (UGTs). UGT1A1 and 1A8 were found to catalyze the formation of both the 6-β- and 4′-β-glucuronides, whereas UGT1A10 formed only the 4′-β-glucuronide. Expressed UGT1A8 catalyzed 6-β-glucuronidation with an apparent Km of 7.9 μM and aVmax of 0.61 nmol/min/mg of protein and 4′-β-glucuronidation with an apparent Kmof 59 μM and a Vmax of 2.0 nmol/min/mg. Kinetic parameters for raloxifene glucuronidation by expressed UGT1A1 could not be determined due to limited substrate solubility. Based on rates of raloxifene glucuronidation and known extrahepatic expression, UGT1A8 and 1A10 appear to be primary contributors to raloxifene glucuronidation in human jejunum microsomes. For human liver microsomes, the variability of 6-β- and 4′-β-glucuronide formation was 3- and 4-fold, respectively. Correlation analyses revealed that UGT1A1 was responsible for 6-β- but not 4′-β-glucuronidation in liver. Treatment of expressed UGTs with alamethicin resulted in minor increases in enzyme activity, whereas in human intestinal microsomes, maximal increases of 8-fold for the 6-glucuronide and 9-fold for the 4′-glucuronide were observed. Intrinsic clearance values in intestinal microsomes were 17 μl/min/mg for the 6-glucuronide and 95 μl/min/mg for the 4′-isomer. The corresponding values for liver microsomes were significantly lower, indicating that intestinal glucuronidation may be a significant contributor to the presystemic clearance of raloxifene in vivo.
Footnotes
- Abbreviations used are::
- UGT
- UDP-glucuronosyltransferase
- R-4-G
- raloxifene-4′-β-glucuronide
- R-6-G
- raloxifene-6-β-glucuronide
- ala
- alamethicin
- rt
- retention time
- MS/MS
- tandem mass spectrometry
- NMR
- nuclear magnetic resonance spectroscopy
- COSY
- proton-proton correlation spectroscopy
- NOE
- nuclear Overhauser enhancement
- LC/MS
- liquid chromatography mass spectrometry
- HPLC
- high-performance liquid chromatography
- UDPGA
- uridine 5′-diphosphoglucuronic acid
- Received December 13, 2001.
- Accepted March 5, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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