Tamoxifen Metabolites Research Articles

The effect of a tamoxifen dose increase from 20 mg to 40 mg in patients with at least one inactive CYP2D6 variant allele and/or concomitant use of a CYP2D6 inhibitor.

584 Background: Genetic polymorphisms leading to reduced CYP2D6 activity and the use of CYP2D6 inhibitors both influence the formation of endoxifen (active metabolite of tamoxifen (TAM)). CYP2D6 genotyping is not part of the standard clinical management of patients treated with TAM and there are no formal recommendations for dose adjustments in patients using TAM and a CYP2D6 inhibitor. We hypothesized that in patients who are either a CYP2D6 intermediate or poor metabolizer and/or are treated with a CYP2D6 inhibitor a dose adjustment of TAM compensates for reduced formation of endoxifen. Methods: Female breast cancer patients treated with TAM 20mg QD for at least 4 weeks were included. They were genotyped for CYP2D6 variant alleles (inactive alleles *3, *4, *5, *6 and decreased activity alleles *10, *41). Patients with at least one inactive allele, two decreased CYP2D6 activity alleles, and/or concomitant use of a CYP2D6 inhibitor received a dose increase of TAM to 40mg QD for 4 weeks. TAM, 4-OH-TAM, N-desmethylTAM and endoxifen levels were determined with LC-MS in blood samples at screening and after 4 weeks. Results: 40 patients with median age 55 (range 37-81) years were enrolled of which 22 extensive (EM), 15 intermediate (IM) and 3 poor metabolizers (PM) for CYP2D6. Three EMs concomitantly used a CYP2D6 inhibitor (paroxetine, escitalopram or citalopram) and were categorized as PM. Mean (SD) endoxifen levels in EMs, IMs, and PMs were 11.4 (5.2), 8.7 (4.6) and 4.1 (1.4) ng/mL, respectively (one-way ANOVA: p=0.007). Three patients were genotyped as *4/*41 or *41/*41, and by consensus classified as IMs; their endoxifen levels (1.9; 3.4; 3.7 ng/mL, respectively) were, however, not different from those observed in PMs. 10 patients have completed the 4 week dose increase (8 IMs, 2 PMs). Mean endoxifen level for the IMs increased from 10.3 to 17.0 ng/mL and from 3.0 to 5.4 ng/mL in the PMs. No serious adverse events occurred after the dose increase. Conclusions: Increasing the dose of TAM from 20 mg to 40 mg QD can compensate for the reduced endoxifen level in CYP2D6 IMs. In contrast, a dose increase to 40mg QD in PMs does not appear to be sufficient to reach endoxifen levels seen in EMs.

Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen and 4′-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 μL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4′-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4′-hydroxytamoxifen and N-desmethyl-4′-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen and 4′-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.

Abstract 4460: Aromatase inhibition by active tamoxifen metabolites

Abstract Introduction: The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent antagonists at the estrogen receptors. Despite this, few data have been published that relate the concentrations of these metabolites to their expected clinical effects. Since anti-estrogenic clinical effects may be brought about not only by estrogen antagonism but also by reduced estrogen concentrations, we tested the direct effects of tamoxifen and its active metabolites on aromatase activity. Methods: The activity of recombinant human aromatase was measured by following the generation of fluorescent metabolite from 7-methoxy-4-trifluoromethylcoumarin (MFC) in microsomal incubations in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen and 4-hydroxy-tamoxifen. Inhibition of aromatase was determined at enzyme concentrations set at 7.5 nM by measuring the percentage of enzyme activity remaining. Results: With MFC concentrations at the Km (25 µM), the IC50s for endoxifen and N-desmethyl-tamoxifen were 6.1 µM and 20.7 µM respectively. At concentrations up to 50 µM, no appreciable inhibition by tamoxifen or 4-hydroxy-tamoxifen was observed. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen across a range of substrate concentrations demonstrated non-competitive kinetics for both inhibitors. The Ki values for endoxifen and N-desmethyl-tamoxifen were 4.0 µM and 15.9 µM respectively. Preincubation of these tamoxifen metabolites with aromatase did not demonstrate any irreversible effect on enzyme activity. Discussion: Among tamoxifen and its three major metabolites, only endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. This enzyme is the major source of estrogen in humans, and changes in its activity may thus alter estrogen-regulated physiology. Inhibition by these tamoxifen metabolites may contribute to changes in serum and tissue estrogen concentrations, and may explain in part the variability in bone density, serum lipid concentration, severity of musculoskeletal pain and breast cancer recurrence observed in patients taking tamoxifen. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and mechanisms that underlie the multiple clinical effects of tamoxifen include the possibility of aromatase inhibition by endoxifen and N-desmethyl-tamoxifen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2011-4460

Abstract 5566: In vitro human skin permeation enhancement of endoxifen by oleic acid

Abstract Background: Oral medication for breast cancer prevention is associated with systemic exposure and resulting adverse effects; there is a need for alternative drug delivery approaches. Transdermal drug delivery is such an option, and 4-Hydroxy-N-desmethytamoxifen (endoxifen, ENX) is an excellent candidate for development for this route. ENX is an active metabolite of tamoxifen with major therapeutic effect related to potent estrogen antagonism and ability to cause proteosomic degradation of ERα. We hypothesize that skin permeation of ENX with oleic acid (OA) will be equivalent or superior to that of estradiol (E2), which is a well-established transdermal agent. Methods: Split-thickness human skin samples (354 ± 25 micron) were prepared from de-identified fresh mastectomy specimens under an IRB approved protocol using a surgical blade and placed in Franz diffusion cells (PermeGear, Inc.). ENX was prepared in 60 % (v/v) ethanol/phosphate buffer (PB) +/- OA (0.1to 1% v/v). E2 was used as a positive control drug, prepared in 60 % ethanol/PB. The receiver fluid was PBS, pH 7.4 with 4% (w/v) polyoxyethylene 20 oleyl ether (Sigma-Aldrich). 100 µL of the drug solution (30 µg + 0.1µCi of 3H-E2 or 3H-ENX) was loaded in the donor chamber. Receiver fluid was stirred at 37° C. A sample of receiver fluid was collected for 3H counting from 4 to 24 hr point with 2 hr interval. Each permeation experiment was performed in duplicate for each treatment condition using the breast skin of one individual. The permeation experiments were repeated three times with the skin from different individuals. Results: Results were expressed as the mean and standard deviation (SD) of the % of the applied dose. Absorption is defined as the amount of drug passing into the receiver fluid. We found that the absorption of E2 was 0.2 ± 0.17% (at 12hr) and 3.16 ± 2.79 % (at 24hr) of the applied dose while the absorption of ENX was 0.07± 0.08 % (at 12hr) and 1.45 ± 1.07 % (at 24hr). In the presence of OA, the permeation of ENX was enhanced by 2 to 12- fold at 12 hr and by 1.2 to 3.8- fold at 24 hr. At the 12 hr point, the absorption of ENX alone is one third of that of E2 alone (p = 0.09) however in the presence of 0.25, 0.5, and 1% (v/v) of OA, the absorption of ENX was by 4.4-fold (p = 0.02), 6.4-fold (p = 0.01), and 3-fold (p = 0.02) higher than E2, respectively. At 24 hr, there was no further significant enhancement in the absorption of ENX by 0.25 and 0.5% of OA compared to that of E2. The addition of 0.1 and 1% OA made the absorption of ENX retarded than that of E2 at 24 hr. Conclusion: The skin permeation of ENX was significantly enhanced in the presence of 0.25-1 % of oleic acid and was superior to that of E2 alone at 12 hr. Our results imply that the topical delivery of endoxifen is a potential option for breast cancer prevention with no or minimal systemic toxicity/side effects. Efficacy remains to be demonstrated, but is highly likely given the data on the parent compound (tamoxifen). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2011-5566

Abstract 3783: Molecular targeting of sphingosine kinase increases the efficacy of hormonal therapy for breast cancer by blocking the catabolic function of autophagy

Abstract In our past studies, we have implicated autophagy as a mechanism of cell survival that contributes to the development of antiestrogen resistance in breast cancer cells (Autophagy. 2009 Apr; 5(3): 400-3). The breast cancer model system used to establish this link between autophagy and antiestrogen resistance was the estrogen-receptor positive MCF-7 cell line. We subjected this cell line to a step-wise selection in increasing concentrations of 4-hydroxytamoxifen (active metabolite of tamoxifen) in the absence of clonal selection and established the antiestrogen resistant TR5 cell line. Analysis of antiestrogen resistant TR5 cells showed induction of ceramide levels during the drug selection that correlated with autophagy induction. Thus, we have begun to characterize the role of key enzymes of the ceramide pathway in pro-survival autophagy relative to the development of antiestrogen resistance. In this study, we show that the inhibition of sphingosine kinase activity effectively kills antiestrogen-sensitive MCF-7 cells and antiestrogen-resistant TR5 cells. Specifically, blockade of spingosine kinase action in MCF-7 and TR5 cells reduced cell number, increased the percentage of cells in the population that showed uptake of trypan blue, induced caspase-9 activation and increased the cleavage of poly-ADP-ribose polymerase and lamin A. Thus, blockade of sphingosine kinase resulted in a robust induction of apoptotic cell death. SIP, the product of sphingosine kinase 1, did not appear to block the death-promoting action of sphingosine kinase blockade. Thus, the pro-survival action(s) of SIP did not compensate for loss of sphingosine kinase action in hormonally-treated breast cancer cells. Importantly, blockade of sphingosine kinase in both MCF-7 and TR5 cells blocked autophagic flux with a marked accumulation of the autophagy proteins LC3 and p62 which are normally turned-over in active autolysosomes. Blockade of autophagic flux was determined using the long-lived protein turnover assay. Although autophagic flux was impaired, autophagosomes were readily seen in the cytoplasm of cells and the lipidated LC3 protein was localized within the membrane of these autophagosomes. Further, studies utilizing electron microscopy to examine cells treated with sphingosine kinase inhibitors did not readily show impaired autophagosome-lysosome fusion. These studies provide strong evidence that sphingosine kinase activity is required for an autolysosomal function downstream of autophagosome-to-lysosome fusion. These studies also support the approach of targeting sphingosine kinase (1 and/or 2) as an effective means to block pro-survival autophagy in breast cancer cells undergoing antiestrogen therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2011-3783

Impact of CYP2D6, CYP3A5, CYP2C9 and CYP2C19 polymorphisms on tamoxifen pharmacokinetics in Asian breast cancer patients

To investigate the impact of genetic polymorphisms in CYP2D6, CYP3A5, CYP2C9 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Asian breast cancer patients. A total of 165 Asian breast cancer patients receiving 20 mg tamoxifen daily and 228 healthy Asian subjects (Chinese, Malay and Indian; n= 76 each) were recruited. The steady-state plasma concentrations of tamoxifen and its metabolites were quantified using high-performance liquid chromatography. The CYP2D6 polymorphisms were genotyped using the INFINITI™ CYP450 2D6I assay, while the polymorphisms in CYP3A5, CYP2C9 and CYP2C19 were determined via direct sequencing. The polymorphisms, CYP2D6*5 and *10, were significantly associated with lower endoxifen and higher N-desmethyltamoxifen (NDM) concentrations. Patients who were *1/*1 carriers exhibited 2.4- to 2.6-fold higher endoxifen concentrations and 1.9- to 2.1-fold lower NDM concentrations than either *10/*10 or *5/*10 carriers (P < 0.001). Similarly, the endoxifen concentrations were found to be 1.8- to 2.6-times higher in *1/*5 or *1/*10 carriers compared with *10/*10 and *5/*10 carriers (P≤ 0.001). Similar relationships were observed between the CYP2D6 polymorphisms and metabolic ratios of tamoxifen and its metabolites. No significant associations were observed with regards to the polymorphisms in CYP3A5, CYP2C9 and CYP2C19. The present study in Asian breast cancer patients showed that CYP2D6*5/*10 and *10/*10 genotypes are associated with significantly lower concentrations of the active metabolite of tamoxifen, endoxifen. Identifying such patients before the start of treatment may be useful in optimizing therapy with tamoxifen. The role of CYP3A5, CYP2C9 and CYP2C19 seem to be minor.

Activity Levels of Tamoxifen Metabolites at the Estrogen Receptor and the Impact of Genetic Polymorphisms of Phase I and II Enzymes on Their Concentration Levels in Plasma

The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.

Lack of any association between functionally significant CYP2D6 polymorphisms and clinical outcomes in early breast cancer patients receiving adjuvant tamoxifen treatment

Active metabolites of tamoxifen are formed mainly by the action of cytochrome P450 2D6 (CYP2D6). Since there are controversies regarding associations between CYP2D6 polymorphisms and outcomes among women with early breast cancer (EBC) treated with tamoxifen, the present evaluation of links with clinical outcomes was conducted. We analyzed a total of 716 patients treated with tamoxifen for hormone receptor positive EBC between 2001 and 2005 at the National Cancer Center, Korea. All patients received tamoxifen 20 mg/day for more than 6 months. DNA obtained from whole blood samples was genotyped for CYP2D6 variants associated with reduced (*10, *41) and absent (*5) activity. Of the total of 716 patients, 558 (77.9%) received adjuvant or neoadjuvant chemotherapy prior to the tamoxifen therapy. From the genotyping of CYP2D6, 152 (21.2%) patients were classified as having the wild type (W/W), 376 (52.7%) one variant allele (W/V), and 188 (26.1%) two variant alleles (V/V). Seventy (9.8%) patients experienced disease recurrence with a median follow-up of 5.6 (range, 0.6-10.3) years. Although known prognostic factors, including tumor size, nodal status, Ki67, PgR negativity, and HER2 positivity showed strong associations with the recurrence free survival (RFS) in this population, no significant association with any of the CYP2D6 genetic variants was evident (P = 0.61; hazard ratio [HR] = 1.14; 95% CI 0.68-1.92). This remained the case after subgroup analysis according to different adjuvant treatments. Polymorphisms of CYP2D6 were not associated with clinical outcomes in EBC patients receiving adjuvant tamoxifen treatment.

Tamoxifen Metabolite Concentrations, CYP2D6 Genotype, and Breast Cancer Outcomes

We explored whether breast cancer outcomes are associated with endoxifen and other metabolites of tamoxifen and examined potential correlates of endoxifen concentration levels in serum including cytochrome P450 2D6 (CYP2D6) metabolizer phenotype and body mass index (BMI). Concentration levels of tamoxifen, endoxifen, 4-hydroxytamoxifen (4OH-tamoxifen), and N-desmethyltamoxifen (ND-tamoxifen) were measured from samples taken from 1,370 patients with estrogen receptor (ER)-positive breast cancer who were participating in the Women's Healthy Eating and Living (WHEL) Study. We tested these concentration levels for possible associations with breast cancer outcomes and found that breast cancer outcomes were not associated with the concentration levels of tamoxifen, 4-hydroxytamoxifen, and ND-tamoxifen. For endoxifen, a threshold was identified, with women in the upper four quintiles of endoxifen concentration appearing to have a 26% lower recurrence rate than women in the bottom quintile (hazard ratio (HR) = 0.74; 95% confidence interval (CI), (0.55-1.00)). The predictors of this higher-risk bottom quintile were poor/intermediate metabolizer genotype, higher BMI, and lower tamoxifen concentrations as compared with the mean for the cohort as a whole. This study suggests that there is a minimal concentration threshold above which endoxifen is effective against the recurrence of breast cancer and that ~80% of tamoxifen takers attain this threshold.

Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen

IntroductionWe have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERβ in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERβ and examine its effectiveness as an anti-estrogenic agent in these cell lines.MethodsMCF7, Hs578T and U2OS cells were stably transfected with full-length ERβ. ERβ protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERβ-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes.ResultsOur data demonstrate that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERβ is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERβ, whereas much higher endoxifen concentrations are needed when ERβ is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERβ to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations.ConclusionsTaken together, these data demonstrate that the presence of ERβ enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/β heterodimers. These findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERβ expression.

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

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 15.9μM, 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.

P-Glycoprotein (ABCB1) Transports the Primary Active Tamoxifen Metabolites Endoxifen and 4-Hydroxytamoxifen and Restricts Their Brain Penetration

P-glycoprotein (P-gp, ABCB1) is a highly efficient drug efflux pump expressed in brain, liver, and small intestine, but also in tumor cells, that affects pharmacokinetics and confers therapy resistance for many anticancer drugs. The aim of this study was to investigate the impact of P-gp on tamoxifen and its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. We used in vitro transport assays and Abcb1a/1b(-/-) mice to investigate the impact of P-gp on the oral availability and brain penetration of tamoxifen and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral administration of tamoxifen (50 mg/kg) was not changed in the absence of P-gp. However, brain accumulation of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were modestly, but significantly (1.5- to 2-fold), increased. Endoxifen, however, displayed a 9-fold higher brain penetration at 4 h after administration. Endoxifen was transported by P-gp in vitro. Upon direct oral administration of endoxifen (20 mg/kg), systemic exposure was slightly decreased in Abcb1a/1b(-/-) mice, but brain accumulation of endoxifen was dramatically increased (up to 23-fold at 4 h after administration). Shortly after high-dose intravenous administration (5 or 20 mg/kg), endoxifen brain accumulation was increased only 2-fold in Abcb1a/1b(-/-) mice compared with wild-type mice, suggesting a partial saturation of P-gp at the blood-brain barrier. Endoxifen, the clinically most relevant metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo, where P-gp limits its brain penetration. P-gp might thus be relevant for tamoxifen/endoxifen resistance of P-gp-positive breast cancer and tumors positioned behind a functional blood-brain barrier.

Investigational study of tamoxifen phase I metabolites using chromatographic and spectroscopic analytical techniques

A comprehensive overview is presented of currently known phase I metabolites of tamoxifen consisting of their systematic name and molecular structure. Reference standards are utilized to elucidate the MS n fragmentation patterns of these metabolites using a linear ion trap mass spectrometer. UV-absorption spectra are recorded and absorption maxima are defined. Serum extracts from ten breast cancer patients receiving 40 mg tamoxifen once daily were qualitatively analyzed for tamoxifen phase I metabolites using a liquid chromatography–tandem mass spectrometry set-up. In total, 19 metabolites have been identified in these serum samples. Additionally a synthetic method for the preparation of the putative metabolite 3′,4′-dihydroxytamoxifen is described.

CYP2D6 Inhibition and Breast Cancer Recurrence in a Population-Based Study in Denmark

Cytochrome P450 2D6 (CYP2D6) inhibition reduces the concentration of 4-hydroxylated tamoxifen metabolites, but the clinical relevance remains uncertain. We conducted a large case-control study nested in the population of 11 251 women aged 35-69 years at diagnosis of stage I-III breast cancer between 1985 and 2001 on Denmark's Jutland Peninsula and registered with the Danish Breast Cancer Cooperative Group. We identified 541 recurrent or contralateral breast cancers among women with estrogen receptor-positive (ER+) disease treated with tamoxifen for at least 1 year and 300 cancers in women with ER-negative (ER-) disease never treated with tamoxifen. We matched one control subject per case patient on ER status, menopausal status, stage, calendar time, and county, genotyped the CYP2D6*4 allele to assess genetic inhibition, and ascertained prescription history to assess drug-drug inhibition. We estimated the odds ratio (OR), associating CYP2D6 inhibition with breast cancer recurrence and adjusted for potential confounding with logistic regression. To address bias from incomplete information on CYP2D6 function, we used Monte Carlo simulation to complete a record-level probabilistic bias analysis. All statistical tests were two-sided. The frequency of the CYP2D6*4 minor allele was 24% in case patients with ER+ tumors, 23% in case patients with ER- tumors, and 22% each in control subjects with ER+ and ER- tumors. In women with ER+ tumors, the associations of one functional allele with recurrence (OR = 0.99; 95% confidence interval = 0.76 to 1.3) and no functional allele with recurrence (OR = 1.4; 95% confidence interval = 0.84 to 2.3) were near null, as were those for women with ER- tumors. The near-null associations persisted when evaluated by intake of medications, by combining genotype with medication history, in the probabilistic bias analysis, or by restricting the analysis to women with ER expression confirmed by re-assay. The association between CYP2D6 inhibition and recurrence in tamoxifen-treated patients is likely null or small.

Should CYP2D6 inhibitors be administered in conjunction with tamoxifen?

Tamoxifen has been used for the treatment or prevention of recurrence in patients with estrogen receptor-positive breast cancers. Because CYP2D6 is known to be a key enzyme responsible for the generation of an active tamoxifen metabolite, ‘endoxifen’, some studies reported that genetic polymorphisms of CYP2D6 that reduced its enzyme activity or coadministration of CYP2D6 inhibitors were associated with the poor clinical outcomes of breast cancer patients treated with tamoxifen. However, there are some discrepant reports for the association between CYP2D6 genotype and clinical outcomes of tamoxifen therapy, probably because of the heterogeneity in sample collection or analysis, including differences in regimen of tamoxifen treatment. A review of published reports regarding the effects of selective serotonin reuptake inhibitors on the pharmacokinetics and/or efficacy of tamoxifen found that concurrent use of strong CYP2D6 inhibitors, especially paroxetine, and possibly others, should be avoided in patients receiving tamoxifen therapy, but there has not been enough evidence for the effects of weak or moderate inhibitors.

Abstract PD05-09: Prospective Assessment of CYP2D6 by Genotyping, Phenotyping and Measurement of Tamoxifen, 4-Hydroxy-Tamoxifen and Endoxifen in Breast Cancer Patients Treated with Tamoxifen

Abstract Background: CYP2D6 is genetically highly polymorphic and several studies support that patients classified as poor-or intermediate-metabolizers achieve less or no benefit from tamoxifen treatment probably because they have lower levels of endoxifen. Genotyping is currently the major method used in studies and in clinical practice. However the ability of genotyping to predict plasma levels of endoxifen is uncertain for an individual patient. We assessed prospectively CYP2D6 activity by genotyping, phenotyping and the measurement of tamoxifen and its metabolites. Methods: Patients were genotyped for CYP2D6 (*1, *3, *4, *5, *6 and *XN) polymorphism. The CYP2D6 phenotype was determined by the dextromethorphan test. Plasma was collected at 2 time points after at least 4 months of treatment with tamoxifen 20 mg daily: tamoxifen, 4-hydroxytamoxifen, N-demethyltamoxifen and endoxifen were measured with high performance liquid chromatography coupled to triple stage tandem mass spectrometry. Linear regression analyses were performed on log transformed concentrations of tamoxifen, its metabolites, and the ratios 4- hydroxytamoxifen/tamoxifen, endoxifen/N-demethyltamoxifen versus the different genotype groups (UM, EM, IM, PM) and the dextromethorphan/dextrorphan ratio. Results: The data of 26 patients are currently available. Geometric mean plasma concentrations (coefficient of variation %) of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen and endoxifen were 377 nmol/L (39%), 482 nmol/L (36%), 5.9 nmol/L (52%) and 60.7 nmol/L (94%), respectively. Genetic variation in CYP2D6 was significantly correlated with endoxifen, 4-hydroxytamoxifen and the ratios of 4-hydroxytamoxifen/tamoxifen and endoxifen/N-demethyltamoxifen: determination coefficients (R-squared) of 44% (P=0.0002), 30% (P=0.0038), 57% (P= &amp;lt;0.0001) and 47% (P=0.0001), respectively. Phenotypes defined by the dextromethorphan/dextrorphan ratio were significantly correlated with endoxifen and the ratios of 4-hydroxytamoxifen/tamoxifen and endoxifen/N-demethyltamoxifen: determination coefficients of 47% (P=0.0002), 39% (P=0.0012) and 59% (P= &amp;lt;0.0001), respectively. Conclusions: Our data confirm a significant correlation between CYP2D6 activity defined by genotyping or by phenotyping and plasma levels of endoxifen. However, the important interindividual variability in the concentrations of the metabolites of tamoxifen remains largely unexplained, suggesting that CYP2D6 genotyping and phenotyping are not sufficient to predict plasma levels of the active metabolites of tamoxifen. These preliminary results are consistent with a potential superiority of monitoring the active metabolites themselves rather than genetic or phenotypic surrogates. The study is ongoing and more data will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-09.

Abstract P5-11-04: Phenotype-Genotype Correlations in Breast Cancer Patients Treated with Tamoxifen

Abstract Background: Personalized medicine strategies are especially relevant for drugs which show a high inter-individual variability regarding their pharmacokinetic and -dynamic properties. Considerable inter-patient variations have been described regarding the plasma concentrations of tamoxifen and its metabolites. Due to the observation that tamoxifen metabolites can show a high pharmacological activity, tamoxifen metabolism has received considerable attention. The metabolism of tamoxifen is complex resulting in a huge number of different products. Important metabolic transformation reactions include demethylation catalyzed by Cytochrome P450 3A4 and hydroxylation mainly catalyzed by Cytochrome P450 2D6 (CYP2D6). The impact of the CYP2D6 activity, which can be predicted from genetic information, on plasma levels and the related pharmacological effects has controversially been discussed. To get a better understanding of the inter-individual differences of the plasma levels of tamoxifen and some of its metabolites we have used liquid chromatography-tandem mass spectrometry (LC/MS/MS) to screen the plasma samples of 106 breast cancer patients. This method enabled the quantification of tamoxifen as well as a relative quantification of demethyltamoxifen and hydroxylated tamoxifen metabolites. To study the impact of enzyme activity on plasma levels, CYP2D6 genotypes were determined as well. Material and methods: For metabolic profiling 1 ml plasma was treated with a solid phase extraction procedure. Chromatographic separation was accomplished on a reversed-phase column. Analytes were detected by mass spectrometry. For genotyping, the CYP2D6 gene was amplified by a long range PCR followed by a multiplexed PCR to generate specific short amplicons. The amplicons were directly analysed by LC/MS. The information necessary to determine the allelic state were obtained from the measured molecular masses. Results: We determined the CYP2D6 genotype of 106 breast cancer patients treated with tamoxifen. We found 19% “Poor Metabolizers", 51% “Intermediate Metabolizers", 29% “Extensive Metabolizers” and 7% “Ultrarapid Metabolizers”. The observed plasma concentrations of tamoxifen and its metabolites showed a high inter-patient variability For instance, plasma concentrations between 26-307 ng/ml (mean 125 ng/ml) were observed for tamoxifen. We did not find a significant impact of the CYP2D6 status on tamoxifen plasma levels. However, the demethyltamoxifen/tamoxifen as well as the hydroxytamoxifen/tamoxifen ratios appear to be influenced by the CYP2D6 status: the higher the metabolic activity, the higher the hydroxytamoxifen content and the lower the demethyltamoxifen content. Conclusions: We have developed analytical tools, which allow the determination of the content of tamoxifen and its major metabolites in human plasma and the genotyping of the CYP2D6 gene. Phenotypic and genotypic data of 106 patients were acquired. Correlation of the data revealed that the genotype had no significant impact on tamoxifen plasma levels. Nevertheless, patients with a predicted higher CYP2D6 activity showed lower demethyltamoxifen/tamoxifen ratios as well as higher hydroxytamoxifen/tamoxifen ratios. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-11-04.

Abstract PD05-11: Development, Characterization, and Effective In Vitro Treatment of an Endoxifen Resistant Breast Cancer Cell Line

Abstract Background: A major issue surrounding the use of therapeutic drugs for the treatment of breast cancer is the eventual development of resistance. Endoxifen, the most potent tamoxifen metabolite, is being developed as a novel endocrine therapy for the treatment of endocrine responsive breast cancer patients. While numerous studies have investigated the process of tamoxifen resistance, no such data exist regarding the mechanism by which cells develop resistance to endoxifen. Here, we describe the development and characterization of a novel endoxifen resistant MCF7 breast cancer cell line and the identification of a specific treatment to effectively target these resistant cells. Methods: MCF7 cells were chronically exposed to concentrations of endoxifen previously demonstrated to be associated with the greatest reductions in estrogen stimulated proliferation and transcription (1000 nM). Changes in the physiological and molecular properties of these cells were monitored during the course of resistance using a wide range of techniques. Results: Following 15 months of endoxifen exposure, an epithelial to mesenchymal transition (EMT) was induced in MCF7 cells, characterized by loss of E-cadherin expression, up-regulation of fibronectin and vimentin expression and increased responsiveness to TGFβ. Resistant cells exhibit a 7-fold increase in their proliferation rates relative to parental cells and display basal like properties (triple negative) due to silencing of ERα , progesterone receptor and CD24 expression. Resistant cells were confirmed to be estrogen insensitive through the use of cell proliferation and gene expression studies. Wound healing and cell migration assays revealed that resistant cells are highly aggressive with a significant level of metastatic potential. Microarray analysis revealed over 7500 genes to be differentially expressed in the resistant cell line relative to parental cells using a 2-fold cutoff, of which only 52 genes (0.7%) were determined to be up-regulated. Interestingly, a number of the most highly altered genes have previously been implicated in the development of EMT and/or resistance including Twist2, IGF binding protein 5, GATA and β-tubulin. Based on the identification of P-tubulin as the most up-regulated gene in resistant cells, 2-methoxyestradiol (2ME2) was recognized as a candidate drug to specifically target this resistant cell line due to it known roles in blocking tubulin polymerization. Indeed, 2ME2 exposure resulted in the induction of apoptosis and significant cell death in vitro. Conclusions: Chronic exposure of MCF7 cells to high concentrations of endoxifen led to induction of EMT with cells that are basal like, estrogen and SERM insensitive, highly TGFP responsive with significantly increased proliferation rates and metastatic potential relative to parental cells. These findings suggest that the mechanisms of resistance to endoxifen may differ from those observed with long term exposure to tamoxifen and have identified 2ME2 as a potentially successful alternative therapy for endoxifen resistant breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-11.

Abstract P4-02-04: Impact of CYP2D6 Genotype and Side-Effects on Adherence Rates to Tamoxifen in Premenopausal Breast Cancer Patients

Abstract Background: Only few studies have investigated the issue of breast cancer patients’ adherence to tamoxifen therapy and factors influencing adherence behavior. Especially in the context of different CYP2D6 genotypes adherence to tamoxifen has not been extensively studied yet. Variations in the CYP2D6 genotype, as well as patients taking inhibitors of CYP2D6 (e.g. antidepressants) contribute to different side effects and adherence rates to adjuvant tamoxifen. Materials and Methods: 106 premenopausal breast cancer patients who met inclusion criteria were consecutively included in the study at the outpatient unit of the Department of Gynecology, Innsbruck Medical University. Within their routine after care appointment patients completed a comprehensive PRO assessment including the FACT-B/ES, the HADS and a self-report questionnaire on adherence behavior (SMAQ). The multi-method approach comprised the Simplified Medication Adherence Questionnaire, a semi-structured interview, physicians’ ratings and blood levels for tamoxifen metabolites. Additionally, the CYP2D6 genotype was determined in all patients participating in this part of the study. Results: 19% patients were poor metabolizer (PM), 51% intermediate metabolizer (IM), 29% extensive metabolizer (EM) and 7% ultra-rapid metabolizer (UM). Significant group differences with regard to tamoxifen and endoxifen serum concentrations were found between the metabolization groups (p=0.044). UMs had the lowest tamoxifen and highest endoxifen concentrations. Only 3.2% of the patients analyzed had no measurable tamoxifen concentrations in their serum. All non-compilant patients were from the extensive metabolizer group. However, during follow-up 25% (2/6) of patients with UM genotype, 13% (4/30) with EM genotype, 1% (1/50) with IM genotype and 0% (0/19) with the PM genotype discontinued their tamoxifen therapy due to therapy related side effects. In addition anti-depressants were more frequently prescribed in the extensive metabolizer group leading to lower levels of the active metabolite endoxifen. Conclusion: A trend towards higher hot flashes was observed in the EM and UM group. We did not find significant differences in QOL between CYP2D6 metabolizing groups. Trend level significance was found for global QOL (on a descriptive level) physical well-being and emotional well-being. Non-adherence to tamoxifen therapy is more frequent in patients with CYP2D6 EM and UM genotype. These preliminary data may explain part of the current controversy over CYP2D6 genotype predicting response to tamoxifen and suggest that patients most likely to benefit from tamoxifen are paradoxically most likely to stop their tamoxifen therapy prematurely. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-04.

Abstract A70: Randomized, placebo-controlled, phase III trial of low-dose tamoxifen in women with intraepithelial neoplasia

Abstract Rationale: Women with breast IEN have an annual risk of invasive disease equal to 8-10 times the general population, thus representing an important target for chemoprevention. In the NSABP-P1 trial, tamoxifen use at 20 mg/day was associated with an 86% reduction of invasive breast cancer in women with previous 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, the increased risk of endometrial cancer and of venous thromboembolism, have significantly limited its broad use in chemoprevention. To improve the risk-benefit ratio, the use of lower doses of the drug has been proposed. Recent trials from our group have shown that a dose of 5 mg/day does not increase endometrial proliferation and is associated with a decrease of the estrogenic activity of tamoxifen on IGF-I, SHBG and antithrombin-III, with a potential decrease of venous thromboembolic events. Moreover, tamoxifen exhibits a high tissue distribution, so that a dose of 5 mg/day 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 tamoxifen. The SNP CYP2D6*4 (1846G&amp;gt;A) allele accounts for 75% of CYP2D6 poor metabolizer phenotype which have been associated with a higher risk to develop a breast event compared to wildtype (Serrano D et al. Pharmacogenomics J. 2010 Mar 23). Study design and enrollment report: This is a multicenter randomized double-blind placebo-controlled phase III trial with two parallel arms: tamoxifen at daily dose of 5 mg or placebo for a total treatment time of 3 years, to assess the efficacy and the safety of 5 mg/day tamoxifen to reduce breast cancer incidence in women with previous IEN (LIN 2-3 and ER-positive or unknown DIN 1b-3). At present no standard treatment exists to treat these women, particularly in Europe, where tamoxifen is not registered in women with prior DCIS (DIN 2-3). A total of 1400 women will be recruited in 3 years to detect a 50% reduction (Hazard Ratio = 0.5) in the incidence of breast cancer in the tamoxifen arm with an 80% power and a 1-sided 5% alpha level. Secondary endpoints include: incidence of other non-invasive breast disorders (i.e., LIN, ductal atypical hyperplasia), endometrial cancer, clinical bone fractures, cardiovascular events, venous thromboembolic events, and clinically manifest cataract. The pharmacogenetic endpoints include to assess whether CYP2D6 genotype can explain modulation on surrogate biomarkers of tamoxifen efficacy and safety, including circulating IGF-I, hormones, mammographic density, endometrial thickness and hot flashes, but also on clinical events. As of July 27, 2010, 10 enrolment centers have been activated, n=128 women have been screened and n=44 have been randomized. 58 women refused to participate and 26 women were excluded for the following ineligibility reasons: 6 women for use of raloxifene, tamoxifen or other SERMs; 4 women for ER negative primary breast cancer; 3 women for bilateral mastectomy, 2 for previous thromboembolic events, 2 for age limits and 9 for other reasons. No serious adverse events or suspected unexpected serious adverse reactions were registered. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A70.