Clinical pharmacokinetics and pharmacogenetics of tamoxifen and endoxifen

Introduction: Tamoxifen dominates the anti-estrogenic therapy in the early and metastatic breast cancer setting. Tamoxifen has a complex metabolism, being mainly metabolized by CYP2D6 into its 30–100 times more potent metabolite, endoxifen. Recently, a phase I study in which endoxifen as an orally z-endoxifen hydrochloride has been successfully evaluated.

Areas covered: the principal pharmacogenetic and non-genetic differences in the pharmacology of tamoxifen and endoxifen are evaluated. To this end, references from PubMed, Embase or Web of Science, among others, were reviewed As non-genetic factors, important differences and similarities such age, or adherence to tamoxifen therapy are comprehensively illustrated. Additionally, since CYP2D6 genotypes are considered the main limitation of tamoxifen, many studies have investigated the association between the worsened clinical outcomes in patients with non-functional CYP2D6 genotypes. In this review, an overview of the research on this field is presented. Also, a summary describing the literature about individualizing tamoxifen therapy with endoxifen concentrations and its limitations is listed.

Expert opinion: z-endoxifen hydrochloride is only investigated in the metastatic setting, still more research is required before its place in therapeutics is known. Similarly, monitoring tamoxifen efficacy based on endoxifen concentrations might not be overall recommended due to the limited evidence available.     

Topical Endoxifen for Mammographic Density Reduction—A Randomized Controlled Trial

Although breast cancer incidence is increasing, there are few primary preventive initiatives. Tamoxifen can reduce breast cancer incidence but is rarely used for primary prevention due to adverse events and tolerance issues. We tested if endoxifen, a tamoxifen metabolite, applied directly to the skin of the breast, could reduce mammographic density, a proxy for therapy response. Ninety women were randomized to placebo, 10 and 20 mg of topical Z-endoxifen for 6 months. Mammographic density and symptoms were measured at baseline and study exit. Despite a high discontinuation rate, driven by skin rashes, we found a significant mammographic density decrease, a dose-dependent increase in the concentration of plasma Z-endoxifen but no systemic side effects. Topical application of tamoxifen metabolites has the potential to decrease breast cancer incidence without major systemic side effects. However, endoxifen may not be suitable for topical administration and is unlikely to be used for breast cancer prevention.     

Curcumin for the Prevention of Epithelial-Mesenchymal Transition in Endoxifen-Treated MCF-7 Breast Cancer Cells

Background: Curcumin was shown to reduce epithelial-mesenchymal transition (EMT) markers in previous shortterm studies. This study was aimed to investigate the potential of curcumin in the prevention of EMT activation inMCF-7 cells induced by endoxifen. Methods: MCF-7 breast cancer cells were treated with Endoxifen 1000 nM+betaestradiol1 nM with or without curcumin (8.5μM or 17 μM). Cells treated with dimethyl sulfoxide (DMSO) 0.001%were used as negative control. After 8 weeks of continuous treatment, the cells were counted, analyzed for mRNAE-cadherin, vimentin, TGF-β expression, total reactive oxygen species (ROS) and observed for morphological changesusing confocal microscope and transmission electron microscope. Result: MCF-7 cell viability was increased inendoxifen + β-estradiol group. Cell viability was significantly decreased in curcumin 17 μM, but not in curcumin8.5 μM group. Analysis of EMT markers at week 8 indicates that there were increase in vimentin and TGF-β mRNAexpressions, while E-cadherin mRNA expressions and TGF-β1 protein concentrations were shown to decrease. Theresults showed that administration of curcumin in all the dose administered were incapable improving the expressionsof vimentin, TGF-β1 and E-cadherin. There was a decrease in ROS concentration in curcumin treated cells (8.5 μM)while in curcumin 17 μM, ROS concentration was increased. Morphological observation using confocal microscopeand TEM showed the presence of mesenchymal cells and adherens junction. Conclusion: endoxifen treatments foreight weeks resulted in upregulation of EMT markers and changes in morphology of MCF-7 breast cancer cells. Theaddition of curcumin did not prevent the activation of EMT.     

Pharmacological Characterization of 4-hydroxy-N-desmethyl Tamoxifen, a Novel Active Metabolite of Tamoxifen

The antiestrogen tamoxifen is extensively metabolized in patients to form a series of compounds with altered affinity for estrogen receptors (ERs), the primary target of this drug. Furthermore, these metabolites exhibit a range of partial agonist and antagonist activities for ER mediated effects that do not depend directly on their absolute affinity for ERs. Thus, clinical response to tamoxifen therapy is likely to depend on the aggregate effect of these different metabolites resulting from their abundance in the patient, their affinity for the receptors, and their agonist/antagonist profile. A recent study has shown that plasma concentrations of the tamoxifen metabolite 4-hydroxy- N -desmethyl tamoxifen (endoxifen), in patents undergoing tamoxifen therapy, are dependent on the cytochrome p450 (CYP) 206 ge notype of the patient and that medications commonly prescribed to patients on tamoxifen therapy can also inhibit endoxifen production. In this study we characterized the properties of this metabolite with respect to binding to ERs, ability to inhibit estrogen stimulated breast cancer cell proliferation and the regulation of estrogen responsive genes. We demonstrate that endoxifen has essentially equivalent activity to the potent metabolite 4-hydroxy tamoxifen (4-OH-tam) often described as the active metabolite of this drug. Since plasma levels of endoxifen in patients with functional CYP2D6 frequently exceed the levels of 4-OH-tam, it seems likely that endoxifen is at least as important as 4-OH-tam to the overall activity of this drug and suggests that CYP2D6 status and concomitant administration of drugs that inhibit CYP2D6 activity have the potential to affect response to tamoxifen therapy.     

In vivo assessment of the metabolic activity of CYP2D6 diplotypes and alleles

Aims

A prospectively enrolled patient cohort was used to assess whether the prediction of CYP2D6 phenotype activity from genotype data could be improved by reclassification of diplotypes or alleles.

Methods

Three hundred and fifty-five patients receiving tamoxifen 20 mg were genotyped for CYP2D6 and tamoxifen metabolite concentrations were measured. The endoxifen: N-desmethly-tamoxifen metabolic ratio, as a surrogate of CYP2D6 activity, was compared across four diplotypes (EM/IM, EM/PM, IM/IM, IM/PM) that are typically collapsed into an intermediate metabolizer (IM) phenotype. The relative metabolic activity of each allele type (UM, EM, IM, and PM) and each EM and IM allele was estimated for comparison with the activity scores typically assigned, 2, 1, 0.5 and 0, respectively.

Results

Each of the four IM diplotypes have distinct CYP2D6 activity from each other and from the EM and PM phenotype groups (each P < 0.05). Setting the activity of an EM allele at 1.0, the relative activities of a UM, IM and PM allele were 0.85, 0.67 and 0.52, respectively. The activity of the EM alleles were statistically different (P < 0.0001), with the CYP2D6*2 allele (scaled activity = 0.63) closer in activity to an IM than an EM allele. The activity of the IM alleles were also statistically different (P = 0.014).

Conclusion

The current systems for translating CYP2D6 genotype into phenotype are not optimally calibrated, particularly in regards to IM diplotypes and the *2 allele. Additional research is needed to improve the prediction of CYP2D6 activity from genetic data for individualized dosing of CYP2D6 dependent drugs.     

Genotype-guided adjuvant endocrine therapy: new tricks from an old drug?

Evaluation of: Schroth W, Antoniadou L, Fritz P et al. Breast cancer treatment outcome with adjuvant tamoxifen relative to patient Cyp2D6 and Cyp2c19 genotypes. J. Clin. Oncol. 25, 5187–5193 (2007).

Tamoxifen is one of the most commonly used treatments for breast cancer but is not effective in all patients. We review the study by Schroth and colleagues, evaluating the correlation between genotype for CYP2D6 and other enzymes involved in tamoxifen metabolism and breast cancer outcome. This study demonstrates that patients treated with tamoxifen with intermediate and poor metabolism genotypes have a higher risk of relapse, independent of other prognostic factors. Patients who were not treated with tamoxifen had no differences in outcomes based on CYP2D6 status. This study adds to the growing body of literature suggesting that genetic differences in patients can determine the effectiveness of tamoxifen and highlights the need for studies evaluating how genotype can best guide selection of endocrine therapy for breast cancer.     

Effects of CYP2D6 and SULT1A1 genotypes including SULT1A1 gene copy number on tamoxifen metabolism.

BACKGROUND: Tamoxifen is hydroxylated by cytochrome P450 (CYP) 2D6 to the potent metabolites 4-hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam), which are both conjugated by sulphotransferase (SULT)1A1. Clinical studies indicate that CYP2D6 and SULT1A1 genotypes are predictors for treatment response to tamoxifen. Therefore, we examined the relationship between CYP2D6 genotype, SULT1A1 genotype, SULT1A1 copy number and the pharmacokinetics of tamoxifen. PATIENTS AND METHODS: The serum levels of tamoxifen and metabolites of 151 breast cancer patients were measured by high-pressure liquid chromatography-tandem mass spectrometry. The CYP2D6 and SULT1A1 polymorphisms and SULT1A1 copy number were determined by long PCR, PCR-based restriction fragment length polymorphism, DNA sequencing and fluorescence-based PCR. RESULTS: The levels of 4OHtam, 4OHNDtam and N-demethyltamoxifen were associated with CYP2D6 predicted enzymatic activity (P < 0.05). The SULT1A1 genotype or copy number did not influence the levels of tamoxifen and its metabolites. However, the ratios of N-demethyltamoxifen/tamoxifen and N-dedimethyltamoxifen/N-demethyltamoxifen were related to SULT1A1 genotype. CONCLUSION: CYP2D6 and SULT1A1 genotypes may partly explain the wide inter-individual variations in the serum levels of tamoxifen and its metabolites. We propose that therapeutic drug monitoring should be included in studies linking CYP2D6 and SULT1A1 genotypes to clinical outcome.     

Low Z‐4OHtam concentrations are associated with adverse clinical outcome among early stage premenopausal breast cancer patients treated with adjuvant tamoxifen

Low steady‐state levels of active tamoxifen metabolites have been associated with inferior treatment outcomes. In this retrospective analysis of 406 estrogen receptor‐positive breast cancer (BC) patients receiving adjuvant tamoxifen as initial treatment, we have associated our previously reported thresholds for the two active metabolites, Z‐endoxifen and Z‐4‐hydroxy‐tamoxifen (Z‐4OHtam), with treatment outcomes in an independent cohort of BC patients. Among all patients, metabolite levels did not affect survival. However, in the premenopausal subgroup receiving tamoxifen alone (n = 191) we confirmed an inferior BC ‐specific survival in patients with the previously described serum concentration threshold of Z‐4OHtam ≤ 3.26 nm (HR = 2.37, 95% CI = 1.02–5.48, P = 0.039). The ‘dose–response’ survival trend in patients categorized to ordinal concentration cut‐points of Z‐4OHtamoxifen (≤ 3.26, 3.27–8.13, > 8.13 nm) was also replicated (P‐trend log‐rank = 0.048). Z‐endoxifen was not associated with outcome. This is the first study to confirm the association between a published active tamoxifen metabolite threshold and BC outcome in an independent patient cohort. Premenopausal patients receiving 5‐year of tamoxifen alone may benefit from therapeutic drug monitoring to ensure tamoxifen effectiveness.

Abbreviations

BC

breast cancer

BCSS

breast cancer‐specific survival

95% CI

95% confidence interval

ER

estrogen receptor

HER2

human epidermal growth factor receptor‐2

LC‐MS/MS

liquid chromatography‐mass spectrometry/ mass spectrometry

pN

pathological nodal status

pT

pathological tumor size

TDM

therapeutic drug monitoring

Z‐4OHtam

Z‐4‐hydroxy‐tamoxifen

     

Post‐marketing assessment of generic tamoxifen in Brazilian breast cancer patients

Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor‐positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We carried out a post‐marketing surveillance of the generic tamoxifen formulation in current use at INCA, by comparing plasma concentrations of the parent drug and metabolites obtained with the generic vs the reference formulation. Thirty patients participated in an open‐label, bracketed protocol, comprising 3 successive phases of 30‐32 days each: the generic formulation was used in phases 1 and 3 and the reference formulation in phase 2. Two blood samples were collected in the last 4 days of each phase, for LC‐MS/MS quantification of tamoxifen and metabolites in plasma. The median plasma concentrations (ng/mL) for the reference formulation were as follows: tamoxifen, 135.0 (CI 95% 114.2‐155.8); endoxifen, 35.3 (30.0‐40.8); and 4‐hydroxytamoxifen, 4.8 (4.2‐5.4). The endoxifen/tamoxifen plasma concentration ratio was 0.27 (0.21‐0.25). ANOVA detected no statistically significant difference in plasma concentrations of tamoxifen, metabolites or the endoxifen/tamoxifen ratio among the three phases. The genetic component (rGC) of the CYP2D6‐mediated conversion of tamoxifen into endoxifen, estimated using the repeated drug administration procedure across the three phases, was 0.87, pointing to an important component of genetic variability. In conclusion, this first post‐marketing surveillance trial of oncologic generic drugs carried out in Brazilian patients verified the switchability between the reference and the generic tamoxifen formulation currently used at our institution. The adopted bracketed protocol adds confidence to this conclusion and may serve as a frame for future trials of post‐marketing assessment of other generic drug products.