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Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer

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Abstract

The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent estrogen receptor antagonists. Attempts to identify relationships between active metabolite concentrations and clinical outcomes have produced mixed results. Since anti-estrogenic effects may be brought about not only by estrogen antagonism, but also by reduced estrogen synthesis, we tested the ability of tamoxifen and its principal metabolites to inhibit aromatase in vitro. The activity of human aromatase in both recombinant and placental microsomal preparations was measured using the rate of generation of a fluorescent metabolite in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen, and Z-4-hydroxy-tamoxifen. Aromatase inhibition was further characterized by measuring the inhibition of testosterone metabolism to estradiol. The biochemical mechanisms of inhibition were documented and their inhibitory potency was compared. Using recombinant human aromatase, endoxifen, and N-desmethyl-tamoxifen were able to inhibit aromatase activity with K i values of 4.0 and 15.9 μM, respectively. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen indicated non-competitive kinetics for both inhibitors. Similarly, endoxifen-inhibited testosterone metabolism via a non-competitive mechanism. No appreciable inhibition by tamoxifen or Z-4-hydroxy-tamoxifen was observed at similar concentrations. The relative inhibitory potency was: endoxifen > N-desmethyl-tamoxifen >>> Z-4-hydroxy-tamoxifen > tamoxifen. Similar data were obtained in human placental microsomes. Endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. Inhibition by these tamoxifen metabolites may contribute to the variability in clinical effects of tamoxifen in patients with breast cancer. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and the biologic mechanisms that underlie these relationships may include aromatase inhibition.

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Abbreviations

Endoxifen:

4-Hydroxy-N-desmethyl-tamoxifen

4HT:

Z-4-hydroxy-tamoxifen

NDMT:

N-desmethyl-tamoxifen

CYP:

Cytochrome P450

CYP19:

Aromatase

MFC:

7-Methoxy-4-trifluoromethylcoumarin

HFC:

7-Hydroxytrifluoromethylcoumarin

HPLC:

High performance liquid chromatography

UV:

Ultraviolet

IC50 :

The half maximal inhibitory concentration

K m :

The Michaelis constant

V max :

The maximum reaction rate

K i :

The equilibrium dissociation constant of the inhibitor

K Sapp :

The apparent Michaelis constant

V maxi :

The apparent maximum reaction rate in the presence of the inhibitor

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Acknowledgments

The authors are grateful to Evan Ogburn MSc for providing technical assistance. This study was supported in part by the National Institutes of Health National Center for Research Resources [Grant K24RR020815] to DF, by the National Institute for General Medical Sciences [Grants T32GM008425, U01GM061373] to DF, and by the Department of Defense Breast Cancer Research Program Predoctoral Fellowship [W81XWH-11-1-0016] to WL.

Conflict of interest

WJL and DAF are authors of a patent submitted to the U.S. patent office that describes new uses of the chemical structures described herein entitled “Materials for inhibiting aromatase and method of using the same to diagnose, treat and monitor breast cancer”.

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Authors and Affiliations

  1. Division of Clinical Pharmacology, Department of Pharmacology and Toxicology, Indiana University Simon Cancer Center, Indiana University School of Medicine, 1001 West 10th Street, Room 7123, Myers Building, Indianapolis, IN, 46202, USA

    Wenjie Jessie Lu

  2. Departments of Medicine, Indiana University Simon Cancer Center, Indiana University School of Medicine, 1001 West 10th Street, Room 7123, Myers Building Indianapolis, Indiana, 46202, USA

    Zeruesenay Desta & David A. Flockhart

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  1. Wenjie Jessie Lu
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  2. Zeruesenay Desta
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  3. David A. Flockhart
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Correspondence to Wenjie Jessie Lu.

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Lu, W.J., Desta, Z. & Flockhart, D.A. Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer. Breast Cancer Res Treat 131, 473–481 (2012). https://doi.org/10.1007/s10549-011-1428-z

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  • DOI: https://doi.org/10.1007/s10549-011-1428-z

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