Tamoxifen Metabolites Research Articles

Abstract P5-05-01: Endoxifen, a novel breast cancer therapy, elicits unique beneficial effects on bone relative to that of other selective estrogen receptor modulators

Abstract Background: Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We and others have previously demonstrated that endoxifen is the most active tamoxifen metabolite responsible for eliciting the anti-cancer effects of this drug and that endoxifen concentrations are an important factor with regard to tamoxifen efficacy. These studies have led to the development of endoxifen as a novel anti-breast cancer drug for which phase I clinical trials are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast gene expression profiles were compared to that of estrogen, tamoxifen, raloxifene and lasofoxifene by microarray and RT-PCR analyses in both estrogen receptor alpha (ERα) and ERβ expressing cell lines. The in vivo effects of an anti-cancer dose of endoxifen (50mg/kg/day) on the skeleton were first analyzed in 3-month-old ovariectomized C57BL/6 mice using Dual-energy X-ray absorptiometry, peripheral Quantitative Computed Tomography, micro-Computed Tomography and histomorphometry. In a second set of studies, a pre-clinical rat model was used to determine the effects of endoxifen (10mg/kg/day) on the skeleton in both a pre- and post-menopausal setting. Results: Endoxifen treatment of ERα and ERβ expressing mouse osteoblast cells led to dramatically different gene expression profiles when compared to that of estrogen and other anti-estrogens. In ovariectomized mice, daily administration of endoxifen led to significant increases in bone mineral density and content throughout the skeleton relative to vehicle control treated animals. The numbers and activity of both osteoblasts and osteoclasts were also found to be significantly higher in endoxifen treated mice. In the pre-clinical model system, endoxifen treatment of 4 month-old ovariectomized Sprague-Dawley rats significantly protected against bone loss following estrogen depletion primarily due to suppression of osteoclast mediated bone resorption. Importantly, in sham operated rats (thus retaining ovarian function), endoxifen treatment enhanced bone volume and trabecular thickness and did not suppress osteoclast activity. Conclusions: These data are the first to examine the effects of the novel breast cancer therapy, endoxifen, on bone and reveal that the molecular mechanisms of action of this compound are substantially different than that of other SERMs. Endoxifen was shown to protect against bone loss following estrogen depletion in both mice and rats and interestingly, enhanced bone mass in ovary intact rats, an observation that is in stark contrast to the known effects of tamoxifen which induces bone loss in the “pre-menopausal” setting. These studies suggest that endoxifen may have superior bone-beneficial effects compared to tamoxifen, and if efficacy is confirmed in later phase trials, endoxifen may represent a better drug of choice for a sub-set of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-01.

Hot flashes are not predictive for serum concentrations of tamoxifen and its metabolites

BackgroundTamoxifen has dramatically reduced the recurrence and mortality rate of estrogen receptor positive breast cancer. However, the efficacy of tamoxifen varies between individuals and 40% of patients will have a recurrence despite adjuvant tamoxifen treatment. Factors that predict tamoxifen efficacy would be helpful for optimizing treatment. Serum concentrations of the active metabolite, endoxifen, may be positively related to treatment outcome. In addition, hot flashes are suggested to be positively associated with tamoxifen treatment outcome.MethodsWe investigated in a series of 109 patients whether the frequency and severity of hot flashes were related to concentrations of tamoxifen and its metabolites. A serum sample of all patients was analyzed for the concentration of tamoxifen, N-desmethyltamoxifen, endoxifen and 4-hydroxytamoxifen, as well as for estradiol concentrations and several single nucleotide polymorphisms in CYP2D6. Additionally, these patients completed a questionnaire concerning biometric data and treatment side effects.ResultsWe found no evidence supporting an association between concentrations of tamoxifen or metabolites and either the frequency or severity of hot flashes in the covariate unadjusted analyses. However, including interactions with menopausal status and pre-treatment hot flash (PTHF) history indicated that post-menopausal women with PTHF experienced an increasing frequency of hot flashes with increasing serum concentrations of tamoxifen and its metabolites. This finding was not altered when adjusting for potential confounding factors (duration of tamoxifen treatment, CYP2D6 phenotype, estradiol serum concentration, age and body mass index). In addition we observed a positive association between body mass index and both hot flash frequency (p = 0.04) and severity (p < 0.0001). We also observed that patients with lower estradiol levels reported more severe hot flashes (p = 0.02).ConclusionsNo univariate associations were observed between concentrations of active tamoxifen metabolites and either the frequency or severity of hot flashes during treatment. However, the frequency of hot flashes may be exacerbated by higher serum concentrations of tamoxifen and its metabolites in post-menopausal women with a history of hot flashes prior to tamoxifen treatment.

The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.

Serum concentrations of tamoxifen and its metabolites increase with age during steady-state treatment

It has been suggested that the concentrations of tamoxifen and its demethylated metabolites increase with age. We measured the serum concentrations of the active tamoxifen metabolites, 4OHtamoxifen (4OHtam), 4-hydroxy-N-desmethyltamoxifen (4OHNDtam, Endoxifen), tamoxifen and its demethylated metabolites. Their relations to age were examined. One hundred fifty-one estrogen receptor and/or progesterone receptor positive breast cancer patients were included. Their median (range) age was 57 (32–85) years. Due to the long half-life of tamoxifen, only patients treated with tamoxifen for at least 80 days were included in the study in order to insure that the patients had reached steady-state drug levels. Tamoxifen and its metabolites were measured by liquid chromatography-tandem mass spectrometry. Their serum concentrations were related to the age of the patients. To circumvent effects of cytochrome (CYP) 2D6 polymorphisms we also examined these correlations exclusively in homozygous extensive metabolizers. The concentrations of 4OHNDtam, tamoxifen, NDtam (N-desmethyltamoxifen), and NDDtam (N-desdimethyltamoxifen) were positively correlated to age (n = 151, p = 0.017, 0.045, 0.011, and 0.001 respectively). When exclusively studying the CYP2D6 homozygous extensive metabolizers (n = 86) the correlation between 4OHNDtam and age increased (p = 0.008). Up to tenfold inter-patient variation in the serum concentrations was observed. The median (inter-patient range) concentration of 4OHNDtam in the age groups 30–49, 50–69, and >69 years were 65 (24–89), 116 (25–141), and 159 (26–185) ng/ml, respectively. We conclude that the serum concentrations of 4OHNDtam (endoxifen), tamoxifen, and its demethylated metabolites increase with age during steady-state tamoxifen treatment. This may represent an additional explanation why studies on the effects of CYP2D6 polymorphisms on outcome in tamoxifen-treated breast cancer patients have been inconsistent. The observed high inter-patient range in serum concentrations of tamoxifen and its metabolites, especially in the highest age group, suggest that use of therapeutic monitoring of tamoxifen and its metabolites is warranted.

Identification of Drug Metabolites in Human Plasma or Serum Integrating Metabolite Prediction, LC–HRMS and Untargeted Data Processing

Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen.

Protoporphyrin IX–β‐Cyclodextrin Bimodal Conjugate: Nanosized Drug Transporter and Potent Phototoxin

Topical or systemic administration of 5-aminolevulinic acid (ALA) and its esters results in increased production and accumulation of protoporphyrin IX (PpIX) in cancerous lesions allowing effective application of photodynamic therapy (PDT). The large concentrations of exogenous ALA practically required to bypass the negative feedback control exerted by heme on enzymatic ALA synthesis and the strong dimerization propensity of ALA are shortcomings of the otherwise attractive PpIX biosynthesis. To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, β-cyclodextrin (βCD) was implemented. The resulting PpIX+βCD product had both carboxylic termini of PpIX connected to the CD. PpIX+βCD was water soluble, was found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines while its phototoxiciy was comparable to that of PpIX. Moreover, PpIX+βCD effectively solubilized the breast cancer drug tamoxifen metabolite N-desmethyltamoxifen (NDMTAM) in water. The PpIX+βCD/NDMTAM complex was readily internalized by both cell lines employed. Furthermore, the multimodal action of PpIX+βCD was demonstrated in MCF7 cells: while it retains the phototoxic profile of PpIX and its fluorescence for imaging purposes, PpIX+βCD can efficiently transport tamoxifen citrate intracellularly and confer cell death through a synergy of photo- and chemotoxicity.

Clinical effects of A4889G and T6235C polymorphisms in cytochrome P-450 CYP1A1 for breast cancer patients treated with tamoxifen: implications for tumor aggressiveness and patient survival

Individual differences in cytochrome P-450 efficiency partly explain their variations in resistance to tamoxifen and estrogen metabolism. Two polymorphisms of the CYP1A1 gene-A4889G and T6235C-are known to affect activation of estrone and estradiol and to deregulate concentration of highly active tamoxifen metabolites. However, the clinicopathologic implications of these findings have not yet been evaluated. The objective of this study is to evaluate whether T6235C and A4889G gene polymorphisms are related to pathological presentations and clinical outcomes of ER+/PR+ breast cancer (BC) in women using tamoxifen. We included 405 women with ER+/PR+ tumors, who used tamoxifen as their primary therapy, and for whom 5-year follow-up data were available. We evaluated associations within clinicopathologic features, including overall 5-year survival, with CYP1A1 gene status. Univariate analysis showed that a slightly higher proportion of women with AG/GG genotypes were of European descent (P=0.05) and that TC/CC genotype was significantly associated with premenopausal status (P=0.01); however, no significant association remained after multivariate adjustment. Women with CYP1A1 genotypes other than AA and TT were more prone to develop low-grade tumors; 85.9% of tumors in AA and TT genotype groups were grade III, but only 76.1% of tumors in carriers of the polymorphisms were grade III (adjusted P=0.02; OR 0.51 for grade III disease; 95% CI 0.28-0.93). After 60months of follow-up, ~75% of the women were alive. There was no significant difference in survival related to the CYP1A1 gene status. Breast cancer patients carrying CYP1A1 gene polymorphisms developed less aggressive tumors, but showed no evidence of better prognoses.

Inhibition of Cytochrome P450 Enzymes by the E- and Z-Isomers of Norendoxifen

Aromatase catalyzes the conversion of testosterone to estradiol and is the main source of endogenous estrogen in postmenopausal women. Aromatase inhibitors (AIs) are used to treat postmenopausal women with hormone receptor-positive breast cancer. Norendoxifen [4-(1-(4-(2-aminoethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenol], an active metabolite of the selective estrogen receptor modulator tamoxifen, has been shown to be a potent competitive AI, with an IC50 of 90 nM. To obtain data relevant to the clinical use of norendoxifen, the primary objective of this study was to investigate norendoxifen's inhibitory capability on enzymes related to drug-drug interactions. We determined the inhibitory ability of norendoxifen against important drug-metabolizing cytochrome P450 enzymes, including CYP1A2, CYP2A6, CYP3A4, CYP3A5, and CYP2C19, to establish the potency of norendoxifen as a potential cause of drug-drug interactions. A second objective was to determine the effects of E- and Z-norendoxifen on the inhibition of these enzymes to further characterize the isomers' selectivity. The inhibitory abilities of E-, mixed, and Z-norendoxifen against recombinant aromatase (CYP19), CYP1A2, CYP3A4, CYP3A5, and CYP2C19 were tested using microsomal incubations. Mixed norendoxifen inhibited these enzymes with Ki values of 70 ± 9, 76 ± 3, 375 ± 6, 829 ± 62, and 0.56 ± 0.02 nM, respectively. E-Norendoxifen had a 9.3-fold-higher inhibitory ability than Z-norendoxifen against CYP19, while E- and Z-norendoxifen had similar potencies against CYP1A2, CYP3A4, CYP3A5, and CYP2C19. These results suggest that norendoxifen is able to act as a potent AI, and that its E-isomer is 9.3-fold more potent than the Z-isomer.

Why Should Results From Metastatic Trials No Longer Matter for Early-Stage Disease?

TO THE EDITOR: The recently published comments by Brauch et al regarding our current knowledge about the importance of CYP2D6 status when prescribing tamoxifen concluded that results from ongoing prospective studies in the metastatic/neoadjuvant setting will not help to close the circle on this topic. We do agree with their conclusion that published retrospective studies—so far—do not provide sufficient evidence to determine whether or not CYP2D6 testing should be performed in daily practice. However, we believe that two registered, ongoing, prospective studies in the neoadjuvant/metastatic setting (the European CYPTAMBRUT-2 study [Study to Assess Response to Tamoxifen in (cT3)/Inoperable Locally Advanced/Metastatic Estrogen Receptor–Positive Breast Cancer by the “Tamoxifen Activity Score” Based on Drug Interaction and Polymorphisms in Genes Coding for Tamoxifen Metabolising Enzymes] and the study of the Eastern Cooperative Oncology Group E3108 [Tamoxifen Citrate in Treating Patients With Metastatic or Recurrent Breast Cancer]) will provide answers that are not available from retrospective studies and which are likely to be relevant in the adjuvant setting. Although metastatic breast cancer differs in many respects from early breast cancer, in which tamoxifen helps to eradicate subclinical disseminated disease, metabolic pathways of tamoxifen for efficacy are disease-stage independent. It is unlikely that endoxifen levels matter in the adjuvant setting if they do not matter in the metastatic setting. However, if endoxifen levels do matter in metastatic disease, it does probably but not necessarily follow that this applies in the adjuvant setting. As principal investigators of the European CYPTAMBRUT-2 study, we would like to stress that this study includes only patients in the first-line metastatic/neoadjuvant setting. Patients with hormoneinsensitive or aromatase inhibitor–refractory disease were excluded from this study, as were premenopausal women. The study has so far recruited 240 of 260 patients. The CYPTAMBRUT-2 study not only addresses response to tamoxifen according to genetic variants but also takes endoxifen levels into account. One should first elucidate the relationship between endoxifen levels and tamoxifen efficacy before investigating the relationship between genetic variants and efficacy. Tamoxifen metabolization is complex; multiple enzymes are involved, and other factors affect endoxifen levels as well. Negative results from studies that have assessed the relationship between genetic variants and efficacy could be a result of the fact that variations in endoxifen levels were not related to efficacy or that the variations in endoxifen levels were not represented well by the analyzed genetic variants. Studies examining the pharmacogenomics of tamoxifen should include the measurement of all relevant metabolites of tamoxifen, as well as all genes and gene polymorphisms that are involved in the metabolism of tamoxifen; in nearly all previous retrospective studies, this has not been the case.

Synthesis of Mixed (E,Z)-, (E)-, and (Z)-Norendoxifen with Dual Aromatase Inhibitory and Estrogen Receptor Modulatory Activities

The first synthesis of the tamoxifen metabolite norendoxifen is reported. This included syntheses of (E)-norendoxifen, (Z)-norendoxifen, and (E,Z)-norendoxifen isomers. (Z)-Norendoxifen displayed affinity for aromatase (Ki 442 nM), estrogen receptor-α (EC50 17 nM), and estrogen receptor-β (EC50 27.5 nM), while the corresponding values for (E)-norendoxifen were aromatase (Ki 48 nM), estrogen receptor-α (EC50 58.7 nM), and estrogen receptor-β (EC50 78.5 nM). Docking and energy minimization studies were performed with (E)-norendoxifen on aromatase, and the results provide a foundation for structure-based drug design. The oral pharmacokinetic parameters for (E,Z)-norendoxifen were determined in mice, and (Z)-norendoxifen was found to result in significantly higher plasma concentrations and exposures (AUC values) than (E)-norendoxifen. The affinities of both isomers for aromatase and the estrogen receptors, as well as the pharmacokinetic results, support the further development of norendoxifen and its analogues for breast cancer treatment.

Synthesis and Biological Activity of 3-N-Substituted Estrogen Derivatives as Breast Cancer Agents

3-N-substituted-estrogen derivatives were synthesized and characterized. Their antiproliferative activities against human ER (+) MCF-7 (Breast), ER (-) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines were determined after 72 hours drug exposure employing CellTiter-Glo assay at concentrations ranging from (0.01-100,000 nM). The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) and raloxifene (RAL). In vitro results indicated that compound 5 (IC50 = 12 µM) displayed comparable antiproliferative activity against MDA-MB 231 cell line; while compounds 6, 7 and 13 (IC50 = 12 µM) displayed higher activity against MCF-7 and Ishikawa cell lines, in comparison to TAM activity (19-33 µM).

CYP2C19*2 predicts substantial tamoxifen benefit in postmenopausal breast cancer patients randomized between adjuvant tamoxifen and no systemic treatment

Estrogen catabolism is a major function of CYP2C19. The effect of CYP2C19 polymorphisms on tamoxifen sensitivity may therefore not only be mediated by a variation in tamoxifen metabolite levels but also by an effect on breast cancer risk and molecular subtype due to variation in lifelong exposure to estrogens. We determined the association between these polymorphisms and tamoxifen sensitivity in the context of a randomized trial, which allows for the discernment of prognosis from prediction. We isolated primary tumor DNA from 535 estrogen receptor-positive, stages I–III, postmenopausal breast cancer patients who had been randomized to tamoxifen (1–3 years) or no adjuvant therapy. Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of CYP2C19*2 and CYP2C19*17. Hazard ratios and interaction terms were calculated using multivariate Cox proportional hazard models, stratified for nodal status. Tamoxifen benefit was not significantly affected by CYP2C19*17. Patients with at least one CYP2C19*2 allele derived significantly more benefit from tamoxifen (HR 0.26; p = 0.001) than patients without a CYP2C19*2 allele (HR 0.68; p = 0.18) (p for interaction 0.04). In control patients, CYP2C19*2 was an adverse prognostic factor. In conclusion, breast cancer patients carrying at least one CYP2C19*2 allele have an adverse prognosis in the absence of adjuvant systemic treatment, which can be substantially improved by adjuvant tamoxifen treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2568-0) contains supplementary material, which is available to authorized users.

A randomized phase II presurgical trial of weekly low-dose tamoxifen versus raloxifene versus placebo in premenopausal women with estrogen receptor-positive breast cancer

IntroductionWe previously demonstrated that 1 or 5 mg per day of tamoxifen (T) given for four weeks before surgery reduces Ki-67 in breast cancer (BC) patients to the same extent as the standard 20 mg/d. Given the long half-life of T, a weekly dose (10 mg per week (w)) may be worth testing. Also, raloxifene (R) has shown Ki-67 reduction in postmenopausal patients in a preoperative setting, but data in premenopausal women are limited. We conducted a randomized trial testing T 10 mg/w vs. R 60 mg/d vs. placebo in a presurgical model.MethodsOut of 204 screened subjects, 57 were not eligible, 22 refused to participate and 125 were included in the study. The participants were all premenopausal women with estrogen receptor-positive BC. They were randomly assigned to either T 10mg/w or R 60 mg/d or placebo for six weeks before surgery. The primary endpoint was tissue change of Ki-67. Secondary endpoints were modulation of estrogen and progesterone receptors and several other circulating biomarkers.ResultsKi-67 was not significantly modulated by either treatment. In contrast, both selective estrogen receptor modulators (SERMs) significantly modulated circulating IGF-I/IGFBP-3 ratio, cholesterol, fibrinogen and antithrombin III. Estradiol was increased with both SERMs. Within the tamoxifen arm, CYP2D6 polymorphism analysis showed a higher concentration of N-desTamoxifen, one of the tamoxifen metabolites, in subjects with reduced CYP2D6 activity. Moreover, a reduction of Ki-67 and a marked increase of sex hormone-binding globulin (SHBG) were observed in the active phenotype.ConclusionsA weekly dose of tamoxifen and a standard dose of raloxifene did not inhibit tumor cell proliferation, measured as Ki-67 expression, in premenopausal BC patients. However, in the tamoxifen arm women with an extensive phenotype for CYP2D6 reached a significant Ki-67 modulation.

CYP2D6 Genotype Should Not Be Used to Determine Endocrine Therapy in Postmenopausal Breast Cancer Patients

The antiestrogen tamoxifen is an effective treatment for all stages of estrogen receptor (ER)-positive breast cancer.1 Tamoxifen blocks estrogen-dependent breast cancer growth by competing with estrogen for binding to its receptor. Tamoxifen itself has antiestrogenic properties, but it is metabolized by a number of cytochrome P450 enzymes into many different metabolites that have varying degrees of antiestrogenic activity. Studies have consistently shown that cytochrome P450 2D6 (CYP2D6), a highly polymorphic drug-metabolizing enzyme, is the enzyme primarily responsible for the production of one of the most potent metabolites, endoxifen (4-hydroxy-N-desmethyl-tamoxifen). Preclinical studies of estrogen-dependent breast cancer have shown that endoxifen is an approximately 100-fold more potent antiestrogen than tamoxifen or the major tamoxifen metabolite, N-desmethyl-tamoxifen. Patients who are homozygous for two null CYP2D6 alleles, and are therefore poor metabolizers (PMs), as well as patients who are taking medications that are CYP2D6 inhibitors, have lower serum endoxifen concentrations as compared with patients who have one or more wild-type CYP2D6 alleles (intermediate metabolizers (IMs) and extensive metabolizers (EMs), respectively) (reviewed by Hertz et al.2).

Prediction of individual tamoxifen activation in breast cancer patients.

e13514 Background: Endoxifen (EN), the main active metabolite of tamoxifen (T), reaches steady state (SS) plasma levels after about 4 months of administration. It derives principally by cytochrome CYP2D6 activity, which is highly variable among patients. In order to predict individual SS EN exposure, three different approaches were investigated. Methods: Before starting T administration, 73 breast cancer patients (median age: 59 yrs, range: 30-89) were characterized by means of: 1) a phenotyping test of CYP2D6 activity, based on the urinary metabolic ratio of dextromethorphan/dextrorphan (log transformed, LMR). 2) CYP2D6 genotyping (alleles *1, *3, *4, *5, *6, *9, *10, *41), to classify patients in 3 functional groups: Extensive (EM), Intermediate (IM) and Poor (PM) Metabolizers. After starting T treatment (20mg/day), EN plasma levels were measured after 1 month (1M EN, pre- steady state) and 4 months of therapy (SS EN). ANOVA, paired and unpaired T test and linear regressions were performed to analyze associations between LMR, CYP2D6 genotype, 1M EN and SS EN. Multivariate linear regression was used to create a predictive equation; its mean prediction and absolute errors (MPE% and MAE%) and the positive and negative predictive values (PPV% and NPV%, according to median SS EN) were calculated. Results: SS EN plasma levels varied between 2.4 and 39.2 (median: 8.7) ng/ml. LMR (median: -1.6; range: -3.1- +1.2) showed a significant linear correlation with SS EN (r=-0.59; p&lt;0.0001). CYP2D6 genotypes (EM=35.6%; IM=50.7%; PM=13.7%) were significantly associated with SS EN (ANOVA, p&lt;0.0001). First month EN plasma levels (median: 5.9 ng/mL; range:1.2-27 ng/ml) were significantly lower than (p&lt;0.0001), and correlated (r=0.87; p&lt;0.0001) with, SS EN. Multiple regression analysis including age, LMR, CYP2D6 genotypes and 1M EN gave the following predictive equation: SS EN= 0.055-1.53 x LMR + 1.11 x 1M EN; (r=0.88; p&lt;0.0001). MPE was 4.1% and MAE was 30%; PPV was 88.5% and NPV was 84.6%. Conclusions: Individual EN exposition at steady state can be reliably predicted by measuring baseline CYP2D6 activity, through the log (dextromethorphan/dextrorphan) metabolic ratio, and EN plasma concentration after 1 month of tamoxifen treatment. Funded by Regione Veneto. Clinical trial information: 532.

Randomized, placebo controlled, phase III trial of low-dose tamoxifen in women with intraepithelial neoplasia.

TPS1610 Background: Breast IntraEpithelial Neoplasia (IEN) is associated with a 8-10 fold increased risk of invasive disease, thus representing an important target for chemoprevention. The NSABP-P1 trial showed that tamoxifen (TAM) at 20 mg/day is associated with a 86% reduction of invasive breast cancer (BC) in women with previous atypical ductal hyperplasia (ADH) (RR=0.14, 95% IC, 0.03-0.47) and with a 56% risk reduction in women with previous LCIS (RR=0.44, 0.16-1.06). However, TAM broad use in chemoprevention has been significantly hampered by the increased risk of endometrial cancer and of venous thromboembolism (VTE). We recently showed that a dose of 5 mg/day does not increase endometrial proliferation, is associated with a decrease of the estrogenic activity of TAM on IGF-I, SHBG and antithrombin-III, and attains at the breast tissue level a concentration 10 times higher than that needed to inhibit cell growth in vitro. The CYP2D6 enzyme mediates oxidation of N-desmethyl tamoxifen to endoxifen, the most active metabolite of TAM. The SNP CYP2D6*4 (1846G&gt;A) allele accounts for 75% of poor metabolizer phenotype which have been associated with a higher risk of a breast event compared to wildtype (Pharmacogenomics J. 2011;11:100-7). Methods: A randomized double-blind placebo-controlled phase III trial to assess the efficacy and safety of 5 mg/day TAM vs. placebo for 3 years in women with previous IEN (LIN 2-3 and ER+ or unknown DIN 1b-3). In Europe TAM is not registered in women with prior DCIS. 1400 women are needed to detect 50% reduction in the incidence of BC in the TAM arm (80% power, 1-sided 5% α error). Secondary endpoints are: incidence of other non-invasive breast disorders, endometrial cancer, clinical bone fractures, cardiovascular events, VTE events, and clinically manifest cataract. Pharmacogenetic endpoints includes the association between CYP2D6 genotype and modulation of biomarkers of TAM efficacy and safety (circulating IGF-I, hormones, mammographic density, endometrial thickness and hot flashes) and clinical events. As of February 5, 2013, 18 enrolment centers have been activated and n=338 women have been randomized. No suspected unexpected serious adverse reactions were registered. Clinical trial information: NCT01357772.

Important and critical scientific aspects in pharmacogenomics analysis: lessons from controversial results of tamoxifen and CYP2D6 studies

Tamoxifen contributes to decreased recurrence and mortality of patients with hormone receptor-positive breast cancer. As this drug is metabolized by phase I and phase II enzymes, the interindividual variations of their enzymatic activity are thought to be associated with individual responses to tamoxifen. Among these enzymes, CYP2D6 is considered to be a rate-limiting enzyme in the generation of endoxifen, a principal active metabolite of tamoxifen, and the genetic polymorphisms of CYP2D6 have been extensively investigated in association with the plasma endoxifen concentrations and clinical outcome of tamoxifen therapy. In addition to CYP2D6, other genetic factors including polymorphisms in various drug-metabolizing enzymes and drug transporters have been implicated to their relations to clinical outcome of tamoxifen therapy, but their effects would be small. Although the results of association studies are controversial, accumulation of the evidence has revealed us the important and critical issues in the tamoxifen pharmacogenomics study, namely the quality of genotyping, the coverage of genetic variations, the criteria for sample collection and the source of DNAs, which are considered to be common problematic issues in pharmacogenomics studies. This review points out common critical issues in pharmacogenomics studies through the lessons we have learned from tamoxifen pharmacogenomics, as well as summarizes the results of pharmacogenomics studies for tamoxifen treatment.

Abstract 4409: The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro.

Abstract Tamoxifen is the most widely used agent in the world for antihormonal therapy of Estrogen Receptor (ER)-positive breast cancer for both pre- and postmenopausal patients. Tamoxifen is a pro-drug and needs to be metabolically activated to a more potent antiestrogen with a higher affinity for the ER. Initially 4-hydroxytamoxifen (4OHT) was the active metabolite of interest, but more recently 4-hydroxy-N-desmethyltamoxifen (endoxifen) is also of significant interest. The role of endoxifen in therapy remains controversial, however both 4OHT and endoxifen exist in two isomeric forms- cis and trans. Previously, it was shown that cis-isomers of tamoxifen metabolites have weak anti-estrogenic properties using in vitro models available (Jordan VC et al, Endocrinology, 1987, 122(4); 1449-54). However, studies show that an increase of cis-isomers of 4OHT in animals and patients may contribute to tamoxifen resistance (Osborne CK et al, JNCI, 1991, 83; 1477-82; Osborne CK et al, J Clin Oncol, 1992, 10; 304-10). Thus, it is important to know the estrogenic properties of clinically relevant tamoxifen metabolites. To document the anti-estrogenic profiles of both geometric isomers of two major hydroxylated metabolites of tamoxifen, isomerically stable fixed-ring derivatives of 4OHT and endoxifen were synthesized. We have assessed the anti-estrogenic properties of these compounds using cell proliferation assays in selectively cloned hypersensitive ER-positive human breast cancer cell lines MCF-7:WS8 and T47D:A18. Also to assess to estrogenic/anti-estrogenic properties of synthesized compounds on transcriptional level, we have used real-time PCR of rat Prolactin (Prl) mRNA isolated from GH3 rat pituitary cell line. Our results indicate that the fixed-ring trans-4OHT and trans-Endoxifen are potent antiestrogens and equivalent to each other, and to their non-fixed-ring counterparts in both cell proliferation assays and Prl gene activity, as they do not possess any agonistic activities by themselves and are able to inhibit the estrogenic actions of 0,1nM 17β-estradiol (E2) completely (IC50 1nM). However, cis-isomers of the fixed-ring compounds are very weakly estrogenic by themselves but are very weak anti-estrogens in combination with 0,1nM E2 in all assays. Conclusions: the weak estrogen-like activity and low potency of the cis-isomers is unlikely to influence the growth of breast cancer during long-term therapy. The high potency of trans-endoxifen and 4OHT will dominate the control of the tumor growth. Support: This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under award number SAC100009 (VCJ). Citation Format: Philipp Y. Maximov, Cynthia Myers, Daphne J. Fernandes, V. Craig Jordan. The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2013-4409

Abstract 5029: The role of estrogen receptor signaling in rhabdomyosarcoma.

Abstract Rhabdomyosarcoma (RMS) is a malignant tumor thought to arise from immature precursors of skeletal muscle cells. In children, RMS accounts for approximately 50% of soft tissue sarcomas and 10% of solid tumors. There are two major histologic subtypes, embryonal and alveolar, both of which exhibit metastatic potential. Despite multimodal therapy (surgery, radiation, and chemotherapy), the failure-free survival of patients with metastatic RMS is only 25%. Currently there is no molecularly targeted therapy for RMS, but several independent clinical observations point to tamoxifen (a selective estrogen receptor modulator) as a promising drug to explore, including the efficacy of tamoxifen against desmoid tumors (which share a similar ontogeny as RMS) and the tendency of RMS to arise during periods of elevated constitutive estrogen exposure. This led our group to investigate estrogen receptor (ER) signaling in RMS. Previous work in our lab has shown that RMS primary tumors and cell lines express ERβ, and that estrogen stimulates RMS cell growth in vitro. In addition, exposure of RMS cells to 4-hydroxytamoxifen (4OHT), an active metabolite of tamoxifen, leads to decreased cell viability and increased apoptotic signaling, accompanied by MAPK activation. These findings suggest that an active ER pathway play a role in RMS, and demonstrate the antagonistic effect of 4OHT in RMS in vitro. To expand on this work, we conducted studies to: 1) further define the molecular mechanism of 4OHT-induced apoptosis in RMS; and 2) test the efficacy of tamoxifen in combination with other chemotherapeutic drugs commonly used to treat RMS (vincristine and actinomycin D). We found that 4OHT (10 μM)-induced apoptosis can be blocked by inhibition of ER and c-Jun N-terminase kinase (JNK). In addition, ER inhibition blocked 4OHT (10 μM)-associated MAPK phosphorylation. Interestingly, long-term treatment (8 weeks) of RMS cells with 4OHT (4 μM) inhibited cell growth as determined by measuring cell population doublings. Using an MTS assay, we found that tamoxifen in combination with vincristine and actinomycin D significantly reduced RMS cell viability compared to vehicle and the drugs alone (p&amp;lt;0.05). These results show that: a) 4OHT-induced apoptosis may be mediated by a signaling sequence from ERs to MAPKs to apoptosis, b) the late growth- inhibitory effect of 4OHT may be caused by both early/transient and late/continuous genes and c) combining tamoxifen with vincristine and actinomycin D is effective against RMS cells in vitro. These findings will guide future gene expression profiling studies, provide a list of specific therapeutic targets that can be tested in vivo, and may find application in clinical protocols in the treatment of RMS. Citation Format: Zainab A. Motala, Kevin Chen, David Malkin. The role of estrogen receptor signaling in rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5029. doi:10.1158/1538-7445.AM2013-5029