Abstract
LGD1069 (Targretin), a retinoid “X” receptor-selective ligand, or rexinoid, is in clinical trials for treating cancer. Biologically-active oxidized LGD1069 metabolites have been observed in patient plasma samples, making corresponding structural characterizations necessary. Formation of multiple metabolite isomersin vivo has created technical challenges in metabolite structural analysis; however, mass spectrometry (MS) was able to pinpoint two sites of Phase I metabolism. A carbon-13 trideuterated analog was used as an isotopic marker to probe Phase II metabolism of LGD1069. Rats were orally gavaged with an equimolar mixture of LGD1069 and [13C2H3]LGD1069, then anesthetized prior to bile-duct cannulation. Bile was collected for 7 hr, extracted, and concentrated. Recovered metabolites were analyzed by narrow-bore, gradient liquid chromatography (LC) with negative ion, electrospray ionization MS detection. When resultant total ion chromatograms were interrogated for mass spectra exhibiting isotope clusters separated by 4 daltons, 13 such clusters corresponding to Phase II LGD1069 metabolites of nine different molecular weights were detected. Acyl-glucuronide and taurine conjugates of both parent compound and hydroxy-LGD1069 were observed. The sulfate and taurine conjugates of oxo-LGD1069 were also identified, as were 6,7-dihydroxy-LGD1069 taurine, LGD1069 ether glucuronide, and a secondary conjugate (taurine) of the latter. Identities of selected conjugates were confirmed by MS/MS. The results of this study demonstrate that when combined with traditional GC/MS and MS/MS data, the isotope cluster technique can provide powerful selectivity in identifying numerous Phase II drug metabolites during a single LC/MS analysis.
Footnotes
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Send reprint requests to: Michael A. Shirley, Ligand Pharmaceuticals, Inc., 10255 Science Center Drive, San Diego, CA 92121.
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Portions of these data were presented in preliminary form at the 7th North American ISSX Meeting, San Diego, Oct. 20–24, 1996, and the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, June 1–5, 1997.
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This work was supported by the National Institutes of Health Grant HL25785.
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↵2 Data on file, Ligand Pharmaceuticals.
- Abbreviations used are::
- HPLC
- high-performance liquid chromatography
- LC
- liquid chromatography
- RAR
- retinoic acid receptor
- RXR
- retinoid “X” receptor
- MS
- mass spectrometry
- PFB-Br
- pentafluorobenzyl bromide
- DIEA
- N,N-diisopropylethylamine
- BSTFA
- bis(trimethylsilyl)trifluoroacetamide
- TMS
- trimethylsilyl
- MOX-HCl
- methoxamine hydrochloride
- ESI
- electrospray ionization
- EI
- electron ionization
- NCI
- negative ion electron capture ionization
- CID
- collision-induced dissociation
- TIC
- total ion chromatogram
- SER
- smooth endoplasmic reticulum
- Da
- daltons
- Received February 26, 1997.
- Accepted June 19, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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