Abstract
A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17β-dihydroexemestane (active metabolite) and 17β-dihydroexemestane-17-O-β-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 μm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1 % aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17β-dihydroexemestane, 17β-dihydroexemestane-d3, 17β-dihydroexemestane-17-O-β-D-glucuronide, and 17β-dihydroexemestane-17-O-β-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4 – 40.0 ng/mL, for exemestane; and 0.2 – 15.0 ng/mL, for 17β-dihydroexemestane and 17β-dihydroexemestane-17-O-β-D-glucuronide, with coefficient of determination (r2) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17β-dihydroexemestane and 92.0 to 103.2% for 17β-dihydroexemestane-17-O-β-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.
Highlights
Breast cancer has remained the most common cancer among females in Singapore over the last forty years.[1]
20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells
Accuracy was calculated as a percentage of the mean value measured over the nominal value at each concentration; precision was expressed in terms of coefficient of variation (CV), defined as percentage of the standard deviation divided by the mean
Summary
Breast cancer has remained the most common cancer among females in Singapore over the last forty years.[1]. [14] In addition, 17DhExe has been reported to be an active metabolite which is subsequently inactivated by glucuronidation to 17β-dihydroexemestane-17-O-β-D-glucuronide (Exe17Oglu). [15, 16] Inactivation of 17DhExe is catalysed by the enzyme UDP-gluconoryltransferase 2B17 (UGT2B17). It has been identified that 60–70% of Asians suffer from homozygous gene deletion of UGT2B17, which can result in reduced glucuronidation of 17DhExe and increased exposure to this active metabolite. [16] simultaneous quantification of Exe, 17DhExe, and Exe17Oglu can aid in determination of the impact of homozygous UGT2B17 gene deletion on the in vivo metabolic profile of Exe. It has been identified that 60–70% of Asians suffer from homozygous gene deletion of UGT2B17, which can result in reduced glucuronidation of 17DhExe and increased exposure to this active metabolite. [16] simultaneous quantification of Exe, 17DhExe, and Exe17Oglu can aid in determination of the impact of homozygous UGT2B17 gene deletion on the in vivo metabolic profile of Exe. [17]
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