Higher Endoxifen Concentrations Research Articles

Clinical CYP2D6 Genotyping to Personalize Adjuvant Tamoxifen Treatment in ER-Positive Breast Cancer Patients: Current Status of a Controversy

Simple SummaryTamoxifen is an important adjuvant endocrine therapy in estrogen receptor (ER)-positive breast cancer patients. It is mainly catalyzed by the enzyme CYP2D6 into the most active metabolite endoxifen. Genetic variation in the CYP2D6 gene influences endoxifen formation and thereby potentially therapy outcome. However, the association between CYP2D6 genotype and clinical outcome on tamoxifen is still under debate, as contradictory outcomes have been published. This review describes the latest insights in both CYP2D6 genotype and endoxifen concentrations, as well CYP2D6 genotype and clinical outcome, from 2018 to 2020.Tamoxifen is a major option for adjuvant endocrine treatment in estrogen receptor (ER) positive breast cancer patients. The conversion of the prodrug tamoxifen into the most active metabolite endoxifen is mainly catalyzed by the enzyme cytochrome P450 2D6 (CYP2D6). Genetic variation in the CYP2D6 gene leads to altered enzyme activity, which influences endoxifen formation and thereby potentially therapy outcome. The association between genetically compromised CYP2D6 activity and low endoxifen plasma concentrations is generally accepted, and it was shown that tamoxifen dose increments in compromised patients resulted in higher endoxifen concentrations. However, the correlation between CYP2D6 genotype and clinical outcome is still under debate. This has led to genotype-based tamoxifen dosing recommendations by the Clinical Pharmacogenetic Implementation Consortium (CPIC) in 2018, whereas in 2019, the European Society of Medical Oncology (ESMO) discouraged the use of CYP2D6 genotyping in clinical practice for tamoxifen therapy. This paper describes the latest developments on CYP2D6 genotyping in relation to endoxifen plasma concentrations and tamoxifen-related clinical outcome. Therefore, we focused on Pharmacogenetic publications from 2018 (CPIC publication) to 2021 in order to shed a light on the current status of this debate.

Abstract P2-11-12: The influences of adherence to tamoxifen and CYP2D6 pharmacogenetics on plasma concentrations of the active metabolite (Z)-endoxifen in breast cancer

Abstract One-third of early breast cancer patients with estrogen receptor (ER) positive tumors treated with the selective estrogen receptor modulator tamoxifen either relapse or die from the disease in the following decade. Improvement of tamoxifen efficacy requires a better knowledge of factors determining outcome of which the medication-taking behavior, i.e. adherence, is a strong suspect. Discontinuation and non-adherence to tamoxifen are frequent and result in increased mortality. Commonly used methods to assess drug adherence such as prescription-refill patterns infer that the medication is taken exactly as prescribed yet, partial adherence or actual use of the medication cannot be controlled. An objective surrogate of tamoxifen adherence is the monitoring of drug concentrations in the patients’ plasma including the parent drug and its active metabolite (Z)-endoxifen (endoxifen), however individual concentrations depend on pharmacogene polymorphisms particularly those of the liver enzyme cytochrome P450 2D6 (CYP2D6). Patients without CYP2D6 variants have normal enzyme function (extensive metabolizer, EM) and achieve high endoxifen concentrations compared to patients with variants conferring absent activity (poor metabolizer, PM) or reduced activity (intermediate metabolizer, IM) who have significantly lower plasma concentrations of the active metabolite. A putative threshold of 5.9 ng/mL endoxifen has been proposed suggesting that patients with endoxifen concentration above or below threshold have lower or higher risk for recurrence, assuming that low endoxifen exposure in the tumor likely results in incomplete inhibition of ER-dependent growth signaling. During tamoxifen treatment it is therefore important that patients achieve clinically relevant endoxifen levels while at the same time avoid confounders of which adherence is a prime candidate. To this end, we investigated patients’ ability to achieve relevant plasma concentrations during tamoxifen treatment in relation to their CYP2D6 metabolizer status and their tamoxifen adherence behavior. Our prospective study included 192 Brazilian breast cancer patients treated with 20 mg tamoxifen daily. Two-thirds of the patients were premenopausal. Plasma levels of tamoxifen and its metabolites were measured by LC-MS/MS at 3, 6 and 12 months of treatment. Adherence to tamoxifen was evaluated by the questionnaire-based Morisky medication adherence scale. CYP2D6 polymorphisms were determined by MALDI-TOF mass spectrometry and real-time PCR. Adherence to tamoxifen decreased from 74% at month 3 to 64% at month 12, and explained 47% of the variability of tamoxifen plasma concentrations (P<.001). Accordingly, plasma monitoring of tamoxifen is useful to inform on tamoxifen adherence. Endoxifen concentrations were associated with CYP2D6 status and adherence. While CYP2D6 explained 26.4% of endoxifen variability at 12 months, the combination with adherence explained 40% (P<.001). The influence of low adherence to not achieving therapeutic endoxifen levels was highest in patients with non-compromised CYP2D6 function (EM phenotype, RR 3.65; 95% CI: 1.48-8.99). we conclude that for patients with intact CYP2D6 function adherence to tamoxifen is critical to achieve therapeutic endoxifen plasma levels. CYP2D6 phenotyping and endoxifen plasma monitoring may help to guide EM (and IM) patients to adhere to tamoxifen which is particularly relevant for premenopausal women who are at higher risk to not adhere to tamoxifen. Citation Format: Jose Claudio Casali da Rocha, Jeanine M Nardin, Werner Schroth, Thais A Almeida, Thomas Mürdter, Solange Picolotto, Evelyn CL Vendramini, Reiner Hoppe, Jenifer P Kogin, Diandra Miqueleto, Silvia DR Moraes, Matthias Schwab, Roberto F Pecoits-Filho, Hiltrud Brauch. The influences of adherence to tamoxifen and CYP2D6 pharmacogenetics on plasma concentrations of the active metabolite (Z)-endoxifen in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-12.

258P - Incidence of clinically significant toxicities in patients with high endoxifen concentrations

258P - Incidence of clinically significant toxicities in patients with high endoxifen concentrations

Comprehensive assessment of cytochromes P450 and transporter genetics with endoxifen concentration during tamoxifen treatment.

Tamoxifen bioactivation to endoxifen is mediated primarily by CYP2D6; however, considerable variability remains unexplained. Our aim was to perform a comprehensive assessment of the effect of genetic variation in tamoxifen-relevant enzymes and transporters on steady-state endoxifen concentrations. Comprehensive genotyping of CYP enzymes and transporters was performed using the iPLEX ADME PGx Pro Panel in 302 tamoxifen-treated breast cancer patients. Predicted activity phenotype for 19 enzymes and transporters were analyzed for univariate association with endoxifen concentration, and then adjusted for CYP2D6 and clinical covariates. In univariate analysis, higher activity of CYP2C8 (regression β=0.22, P=0.020) and CYP2C9 (β=0.20, P=0.04), lower body weight (β=-0.014, P<0.0001), and endoxifen measurement during winter (each β<-0.39, P=0.002) were associated with higher endoxifen concentrations. After adjustment for the CYP2D6 diplotype, weight, and season, CYP2C9 remained significantly associated with higher concentrations (P=0.02), but only increased the overall model R by 1.3%. Our results further support a minor contribution of CYP2C9 genetic variability toward steady-state endoxifen concentrations. Integration of clinician and genetic variables into individualized tamoxifen dosing algorithms would marginally improve their accuracy and potentially enhance tamoxifen treatment outcomes.

CYP2D6 genotypes, endoxifen levels, and disease recurrence in 224 Filipino and Vietnamese women receiving adjuvant tamoxifen for operable breast cancer

BackgroundWhile tamoxifen activity is mainly due to endoxifen and the concentration of this active metabolite is, in part, controlled by CYP2D6 metabolic status, clinical correlative studies have produced mixed results.FindingsIn an exploratory study, we determined the CYP2D6 metabolic status and plasma concentrations of endoxifen among 224 Filipino and Vietnamese women participating in a clinical trial of adjuvant hormonal therapy for operable breast cancer. We further conducted a nested-case–control study among 48 women (half with recurrent disease, half without) investigating the relationship of endoxifen concentrations and recurrence of disease.We found a significant association of reduced endoxifen plasma concentrations with functionally important CYP2D6 genotypes. High endoxifen concentrations were associated with higher risk of recurrence; with a quadratic trend fitted to a stratified Cox proportional hazards regression model, the likelihood ratio p-value was 0.002. The trend also showed that in 8 out of 9 pairs with low endoxifen concentrations, the recurrent case had lower endoxifen levels than the matched control.ConclusionsThis exploratory analysis suggests that there is an optimal range for endoxifen concentrations to achieve favorable effects as adjuvant therapy. In particular, at higher concentrations (>70 ng.ml), endoxifen may promote recurrence.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-52) contains supplementary material, which is available to authorized users.

Abstract PD10-08: Venlafaxine inhibits the CYP2D6 mediated metabolic activation of tamoxifen: Results of a prospective multicenter study: (NCT00667121)

Abstract Background: CYP2D6 is the rate limiting enzyme responsible for the metabolic activation of tamoxifen (tam) to endoxifen. Compared to CYP2D6 poor metabolizers (PM), tam-treated CYP2D6 extensive metabolizers (EM) have higher endoxifen concentrations, more vasomotor symptoms (Goetz, MP J Clin Oncol 2005), and are more likely to discontinue tam (Rae, JM 2009. Pharmacogenomics J). Additionally, higher endoxifen concentrations are associated with a stepwise increase in tam side-effects (Lorizio, W Breast Cancer Res Treat 2012). The data regarding CYP2D6 genotype and recurrence is mixed. Venlafaxine is a weak CYP2D6 inhibitor not known to alter tam pharmacokinetics (PK) and commonly recommended for tam induced hot flashes. We conducted a multicenter pharmacological study to determine whether venlafaxine altered the PK of tam and to determine the distribution of CYP2D6 genotypes in this population Methods: Women taking tam for at least 4 weeks and for whom venlafaxine was recommended for the treatment of hot flashes were eligible. Blood samples were collected prior to and 8–16 weeks following initiation of venlafaxine for steady state tam and metabolites. Genotyping was performed for alleles associated with no (PM; *3, *4, *5,*6); reduced (intermediate, IM; *10, 17 and *41); and ultra-rapid (UM; *1×2) metabolism. Power calculations demonstrated that 17 patients with paired samples were required (two-sided alpha=0.05 t-test, 90% power) to detect a 25% change in endoxifen levels after at least 8 weeks of concurrent treatment. Results: 30 women (median age 48.5) initiated venlafaxine. CYP2D6 genotypes were within Hardy Weinberg Equilibrium (HWE). CYP2D6 UM allele frequency (6.7%) was higher while CYP2D6 *4 (13.3%) was lower than expected compared to an unselected population (0.5 and 21% respectively; Sachse Am. J. Hum. Genet. 1997), resulting in the absence of CYP2D6 PM/PM. Mean (min/max) baseline endoxifen concentrations (8.73; 1.5–20.5 ng/ml) were correlated with CYP2D6 phenotype as follows: intermediate (EM/PM, PM/IM): 6.8 (1.5–11.2); extensive (EM/EM, EM/IM): 9.4 (1.5–20.5) and ultra-rapid (UM/EM: 11.0; 7.8–14) (r2 = 0.35 p = 0.05). In patients with paired samples (n = 20), venlafaxine resulted in a 23% decrease in endoxifen (−2.06 ng/ml; 95% CI −0.69 to −3.04; p = 0.004), but not tam, NDMT, or 4HT concentrations. Following initiation of venlafaxine, CYP2D6 genotype was no longer associated with endoxifen concentrations (r2 = 0.28 p = 0.23). For women with reduced CYP2D6 metabolism [EM/PM (n = 9) or PM/IM (n = 1)], venlafaxine lowered endoxifen concentrations (−2.98 ng/ml) to a level (5.41 ng/ml) reported to be associated with a higher risk of recurrence in adjuvant tam treated patients (Madlensky, L Clin Pharmacol Ther 2011). Conclusions: In this study, women with tam-induced vasomotor symptoms requiring venlafaxine were comprised predominantly of CYP2D6 EM and UM metabolizers. Venlafaxine significantly decreased endoxifen concentrations. Although the optimal concentration of endoxifen is unknown, given prior data linking low endoxifen concentrations with recurrence, venlafaxine should be used with caution in tam treated patients. (Supported by R01CA133049) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-08.

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.

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.

Abstract 3603: Endoxifen pharmacokinetics and bioavailability in female mice

Abstract In collaboration with NCI, we are developing endoxifen (END), the active metabolite of tamoxifen (TAM), as a drug for the treatment of estrogen receptor positive breast cancer. Tamoxifen treated women with reduced or absent CYP2D6 enzyme activity have significantly lower END plasma concentrations. In a retrospective analysis of two different prospective tamoxifen trials (NCCTG and ABCSG), we have demonstrated that genetic or drug-induced reductions in CYP2D6 metabolism are associated with a higher risk of breast cancer recurrence in Tam treated ER positive breast cancer. To test the hypothesis that clinically relevant concentrations and exposures of END are achieved following oral administration of END, we characterized the pharmacokinetics of i.v. and oral END and oral TAM in female mice. Endoxifen was synthesized and provided by NCI/DTP. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. Following i.v. administration of a 1 mg/kg dose, peak plasma concentration, terminal elimination half-life and plasma clearance values were 0.26 µM, 6.5 h and 11.8 L/h/kg, respectively. Oral pharmacokinetics and bioavailability were determined following doses of 50 and 200 mg/kg. Peak plasma concentrations of 0.76 µM were achieved with a 50 mg/kg dose and plasma concentrations above 0.1 µM were maintained for longer than 8 h. Peak plasma concentrations of 8 µM were achieved with a 200 mg/kg dose and plasma concentrations above 2 µM were maintained for 24 h. Bioavailability was 50% and greater than 100% following the 50 mg/kg and 200 mg/kg doses, respectively. Based on pharmacokinetic data for oral END and TAM normalized to a 50 mg/kg oral dose, END plasma concentrations and total exposure were 20-fold greater following oral END as compared to oral TAM. In conclusion, END has high oral bioavailability in mice and substantial plasma concentrations are achieved and maintained after a single oral END dose. Compared to an equivalent oral dose of Tam, substantially higher END concentrations are achieved following oral END. These data suggest that the primary administration of End may overcome the limitations related to human CYP2D6 metabolism, and support the ongoing development of endoxifen as a primary therapy for ER breast cancer. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3603.

Endoxifen, but not 4-hydroxytamoxifen, degrades the estrogen receptor in breast cancer cells: a differential mechanism of action potentially explaining CYP2D6 effect.

Abstract Abstract #19 Background: Tamoxifen (TAM) is a standard endocrine therapy for the treatment of women with estrogen receptor (ER) positive breast cancer. TAM is activated by the cytochrome P450 2D6 enzyme system into two potent and active metabolites, 4-hydroxytamoxifen (4HT) and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). While human concentrations of 4HT are negligible and vary little in plasma (5-10 nM), endoxifen concentrations vary widely (10-180 nm), and women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. Despite these observations, endoxifen's contribution to tamoxifen's overall drug effectiveness is uncertain.&amp;#x2028; Methods: Using cells endogenously expressing ERa (MCF7, T47D) and cells stably transfected with ERa (Hs578T and U2OS), we examined the relative effects of TAM and its primary metabolites on ERa protein levels by western blotting, ERa transcriptional activity by luciferase reporter assays and real-time RT-PCR, and on ER positive breast cancer cell growth through the use of proliferation assays.&amp;#x2028; Results: We have discovered that endoxifen induces ERa protein turnover through proteasomal degradation similar to that of ICI in a concentration and time-dependent manner. These findings are in stark contrast to TAM, N-desmethyl-tamoxifen (NDT) and 4HT, which stabilize the ER. Optimal degradation occurs only at endoxifen concentrations observed in human CYP2D6 extensive metabolizers (&amp;gt; 40 nM) and persists even in the presence of TAM (300 nM), 4HT (7 nM), and NDT (700 nM) at concentrations observed in patients receiving tamoxifen therapy. In contrast, reducing endoxifen concentrations to those observed in a CYP2D6 poor metabolizer (20 nM), without altering TAM, 4HT, and NDT, results in ER stabilization. High endoxifen concentrations (100-1000 nM) completely block estrogen (E2)-induced ER transcriptional activity even in the presence of TAM, 4HT, and NDT, while low endoxifen concentrations (20-40 nM) do not. Further, low concentrations of endoxifen (20 nM) do not significantly alter E2-induced cell proliferation; however, high concentrations of endoxifen (100-1000 nM) completely block this process. Discussion: Our data demonstrate that endoxifen is a potent anti-estrogen that targets ERa for proteasomal degradation, blocks ERa transcriptional activity and inhibits E2-induced breast cancer cell proliferation. Importantly, these effects of endoxifen are observed at concentrations found in CYP2D6 extensive metabolizers and are maintained even in the presence of TAM, 4HT and NDT. These studies suggest that endoxifen may be the primary metabolite responsible for tamoxifen's effectiveness in the treatment of ER positive breast cancer and provide the impetus to evaluate whether TAM-related side-effects, such as thrombo-embolism and endometrial hyperplasia/carcinoma, are inversely associated with a patient's ability to metabolically activate tamoxifen. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 19.

Pharmacogenetic variants influence tamoxifen's estrogenic effect on bone density

Background Long-term effects from tamoxifen therapy for breast cancer include changes in bone mineral density (BMD). Methods We examined the effects of menopausal status, tamoxifen and its metabolite concentrations on BMD in 69 women treated with 20mg of tamoxifen for one year. We tested for associations between genetic variants in CYP2D6, Estrogen Receptor α (ER) and change in BMD, measured in the lumbar spine and hip. Variants in CYP2D6 (*3, *4, *5, *6 and *10) and estrogen receptor (PvuII and XbaI) were identified by RFLP assays. Results We noted a significant decrease in lumbar BMD in pre-menopausal women (Mean −0.069± 0.057 g/cm2, p=.0001). No significant changes in post-menopausal women were found (p=.27). Pre-menopausal women with non-variant CYP2D6 genotypes experienced greater decrease in lumbar BMD than those who were *1/*4 or *4/*4 (p=.041). The active tamoxifen metabolite endoxifen was significantly correlated with lumbar spinal change in this pre-menopausal group (r2= 0.22, p= 0.023). ER variants did not predict these changes in BMD. In post-menopausal women who carried the ERα PvuII TT genotype there was a significant increase in hip BMD (Mean +0.019± 0.050 g/cm2, p=.023). Conclusion Pre-menopausal women experienced loss in lumbar spine BMD during tamoxifen treatment that was associated with CYP2D6 wild-type genotype and high endoxifen concentrations. In post-menopausal women, ERα TT genotype was associated with an increase in hip BMD. Clinical Pharmacology & Therapeutics (2005) 77, P21–P21; doi: 10.1016/j.clpt.2004.11.082