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Oxidative metabolism of a rexinoid and rapid phase II metabolite identification by mass spectrometry
M A Shirley1, P Wheelan, S R Howell
1Department of Drug Safety and Disposition, Ligand Pharmaceuticals, Inc., San Diego, CA 92121, USA.
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 isomers in 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.
Insights
Researchers identified numerous Phase II metabolites of LGD1069, a cancer drug candidate, using an isotopic labeling technique. This method aids in characterizing complex drug metabolites for improved cancer treatment development.
Area of Science:
- Pharmacology
- Metabolomics
- Analytical Chemistry
Background:
- LGD1069 (Targretin) is a retinoid X receptor-selective ligand (rexinoid) investigated for cancer treatment.
- Observed biologically active oxidized metabolites necessitate structural characterization.
- In vivo formation of multiple metabolite isomers presents analytical challenges.
Purpose of the Study:
- To characterize Phase I and Phase II metabolites of LGD1069.
- To develop and apply a novel isotopic labeling strategy for metabolite identification.
- To enhance the structural analysis of complex drug metabolites.
Main Methods:
- Utilized a carbon-13 trideuterated analog of LGD1069 as an isotopic marker.
- Administered an equimolar mixture of LGD1069 and its labeled analog to rats.
- Collected and analyzed bile samples using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).
- Employed isotope cluster detection to identify Phase II metabolites.
Main Results:
- Identified 13 isotope clusters corresponding to nine different molecular weight Phase II LGD1069 metabolites.
- Detected acyl-glucuronide and taurine conjugates of parent LGD1069 and hydroxy-LGD1069.
- Identified sulfate and taurine conjugates of oxo-LGD1069, dihydroxy-LGD1069 taurine, ether glucuronide, and a secondary taurine conjugate.
- Confirmed identities of selected conjugates using MS/MS.
Conclusions:
- The isotope cluster technique, combined with traditional MS methods, offers powerful selectivity for identifying numerous Phase II drug metabolites.
- This approach facilitates comprehensive metabolite profiling in a single LC/MS analysis.
- The findings contribute to a better understanding of LGD1069 metabolism, crucial for its clinical development in cancer therapy.