Characterization of the isomeric configuration and impurities of (Z)-endoxifen by 2D NMR, high resolution LC⬜MS, and quantitative HPLC analysis

Abstract

(Z)-Endoxifen (4-hydroxy-N-desmethyltamoxifen), an active metabolite generated via actions of CYP3A4/5 and CYP2D6, is a more potent selective estrogen receptor modulator (SERM) than tamoxifen. In the MCF-7 human mammary tumor xenograft model with female athymic mice, (Z)-endoxifen, at an oral dose of 4– 8 mg/kg, significantly inhibits tumor growth. (Z)-Endoxifen's potential as an alternative therapeutic agent independent of CYP2D6 activities, which can vary widely in ER+ breast cancer patients, is being actively evaluated. This paper describes confirmation of the configuration of the active (Z)-isomer through 2D NMR experiments, including NOE (ROESY) to establish spatial proton–proton correlations, and identification of the major impurity as the (E)-isomer in endoxifen drug substance by HPLC/HRMS (HPLC/MS-TOF). Stability of NMR solutions was confirmed by HPLC/UV analysis. For pre-clinical studies, a reverse-phase HPLC–UV method, with methanol/water mobile phases containing 10 mM ammonium formate at pH 4.3, was developed and validated for the accurate quantitation and impurity profiling of drug substance and drug product. Validation included demonstration of linearity, method precision, accuracy, and specificity in the presence of impurities, excipients (for the drug product), and degradation products. Ruggedness and reproducibility of the method were confirmed by collaborative studies between two independent laboratories. The method is being applied for quality control of the API and oral drug product. Kinetic parameters of Z- to E-isomerization were also delineated in drug substance and in aqueous formulation, showing conversion at temperatures above 25 °C.

Introduction of Methyl Groups at C2 and C6 Positions Enhances the Antiangiogenesis Activity of Curcumin

“…The E,E -configurations of the 2,6-dimethyl groups of 1 were confirmed by two-dimensional rotating-frame nuclear Overhauser effect spectroscopy (ROESY) 22 23 24 . These experiments showed no cross-peaks between methyl protons (H a ) at C2 and C6 positions (2.15 ppm) and vinylic protons (H b ) at C1 and C7 positions (7.61 ppm), indicating that these methyl and vinylic protons do not exist in close proximity in space (<5 Å).…”

Preliminary results using a kit to measure tamoxifen and metabolites concentrations in capillary blood samples from women with breast cancer

“…However, unlike the END concentrations, the 4HT concentrations in the capillary blood samples were lower than in venous blood. Given that these are each complex molecules which are capable of reacting in different ways, potentially resulting in different degradation products and rearrangements, we cannot exclude that the peaks interpreted as 4HT and END during LC–MS/MS also had contributions from products that had similar chromatographic retention times to 4HT and END 14 , 15 . Thus, differences in the products of 4HT and END may have also contributed to the differences in the 4HT and END concentrations.…”

Tamoxifen Isomers and Metabolites Exhibit Distinct Affinity and Activity at Cannabinoid Receptors: Potential Scaffold for Drug Development

“…Functionally, Z-Tam acts as an ER antagonist, while E-Tam acts as an ER agonist [23, 24]. Similar differences in affinity and intrinsic activity favoring the Z isomer have also been observed with 4OHT and End [25, 26]. Such distinct modulation of ERs by the E and Z isomers of Tam, 4OHT and End [27] suggest that these isomers might also exhibit novel affinity and activity at CB1 and CB2Rs.…”