Tamoxifen Metabolism Research Articles

Abstract 108: Coffee prevents early events in tamoxifen-treated breast cancer patients and modulates hormone receptor status.

Abstract Whether coffee modulates response to endocrine therapy in breast cancer patients is currently unknown. The CYP1A2 and CYP2C8 enzymes contribute to tamoxifen and caffeine metabolism. The aim was to investigate the impact of coffee consumption on tumor characteristics and disease-free survival in relation to breast cancer treatment and CYP1A2 and CYP2C8 genotypes. Questionnaires regarding lifestyle were completed preoperatively by 592 patients in Southern Sweden. CYP1A2*1F and CYP2C8*3 were genotyped. Clinical data and tumor characteristics were obtained from patients’ charts, population registries, and pathology reports. Coffee consumption was categorized as low (0-1 cups/day), moderate (2-4 cups/day), or high (5+cups/day). The proportion of estrogen receptor negative (ER-) tumors increased with increasing coffee consumption (Ptrend=0.042). Low consumption was associated with higher frequency of discordant receptor status (ER+PgR-) OR 2.61 (1.59-4.29) compared to higher consumption. Median follow-up time was 4.92 years. Tamoxifen-treated patients with low consumption had significantly increased risk for early events compared to patients who consumed two or more cups/day, adjusted HR 2.50 (1.20-5.26). Low coffee consumption was also associated with higher risk for distant metastases in tamoxifen-treated patients with ER+ tumors, adjusted HR 2.43 (1.00-5.88). Low consumption combined with at least one CYP1A2*1F C-allele or CYP2C8*3 was associated with a high risk for early events in tamoxifen-treated patients, adjusted HRs 3.49 (1.54-7.90) and 6.14 (2.46-15.34), respectively. In conclusion, a moderate to high coffee consumption of caffeinated coffee was associated with significantly lower risk for early breast cancer events in tamoxifen-treated patients. This may be due to modification of the hormone receptor status or altered CYP enzyme activity. If confirmed, new recommendations regarding coffee consumption during tamoxifen-treatment may be warranted. Citation Format: Maria Simonsson, Viktoria Söderlind, Maria Henningson, Maria Hietala, Carsten Rose, Christian Ingvar, Helena C. Jernström. Coffee prevents early events in tamoxifen-treated breast cancer patients and modulates hormone receptor status. [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 108. doi:10.1158/1538-7445.AM2013-108

Tamoxifen Benefits and <i>CYP2D6</i> Testing in Women With Hormone Receptor-Positive Breast Cancer

Cancer intervention strategies have been increasingly focused on developing therapies that are personalized and tailored to each individual's unique genetic profile. Evolving understanding of the metabolism and pharmacogenomics of tamoxifen, an early example of targeted therapy for women with hormone receptor-positive breast cancer, has created decision-making challenges for healthcare providers and their patients. This article reviews the pharmacology of tamoxifen, the genetics and physiology of the CYP2D6 enzyme system that has important effects on tamoxifen metabolism, and subset data analyses from large controlled, clinical trials that cast new light on previously held beliefs about the utility of CYP2D6 genotyping for predicting tamoxifen effectiveness and improved breast cancer outcomes in women with early-stage, hormone receptor-positive breast cancer.

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA

A selective and low-cost CD-MEKC method under acidic conditions was developed for investigating the N-oxygenation of tamoxifen (TAM) by flavin-containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N-oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β-CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis-Menten constant (K(m)) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V(max)) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K(i)) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti-tumour agent, were investigated.

Abstract P6-04-05: Tamoxifen dose escalation based on endoxifen level: a prospective trial with genotyping, phenotyping and pharmacokinetics over 4 months.

Abstract Background: Retrospective studies assessing the impact of tamoxifen (Tam) metabolism and its active metabolite, endoxifen, on the efficacy of the treatment produced conflicting results. The prospective CYPTAM-BRUT 2 trial is ongoing1. In the present study we assessed if the level of Tam metabolites could be improved by doubling tamoxifen dose in breast cancer patients (pts) with any CYP2D6 genotype, poor (PM), intermediate (IM) and also extensive metabolizer (EM). Patients and methods: This multicenter, prospective, open-label trial included pts treated with Tam for ≥ 4 months. CYP2D6 activity was determined centrally by genotyping and phenotyping (dextromethorphan test). Liquid chromatography-tandem-mass spectrometry was used to measure Tam, N-desmethyltamoxifen (N-DMT), 4-hydroxytamoxifen (4-HT) and endoxifen twice at baseline (Tam 20 mg qd), then at days 30, 90 and 120 after having increased the dose to 20 mg bid. Endoxifen increase and the differences between genotype/phenotype subgroups were analyzed by ANOVA. Results: 76 pts were analyzed. Steady-state concentrations for Tam and its metabolites were reached in 30 days after doubling the dose. A range of 1.6 to 1.8 fold increase was observed. Geometric mean plasma concentrations in ng/ml (CV%) were: at baseline and day 30 respectively 134 (48) and 246 (46) for tamoxifen (p < 0.0001); 246 (53) and 413 (48) for N-DMT (p < 0.0001); 2.3 (44) and 3.7 (51) for 4HT (p < 0.0001); 18.7 (89) and 31.1 (92) for endoxifen (p = 0.005). The level of endoxifen increased 1.4 to 1.7 folds in all genotype subgroups with geometric mean plasma concentrations in ng/ml (CV%): 6.9 (36) to 9.7 (24) in PMs (p = 0.7); 14.2 (69) to 20.7 (76) in IMs (p < 0.0001); and 22.6 (76) to 38.7 (85) in EMs (p < 0.0001). Similar results were obtained while considering phenotype subgroups. Genotypes and phenotypes explained less than 30% of the variability in endoxifen levels. The occurrence of hot flashes and night sweating were followed prospectively. Endoxifen levels did not predict an increase in HF/NS events' overall occurrence (OR = 1.01, CI95% 0.78–1.31 for HF and 1.01, CI95% 0.79–1.29 for NS). Twelve pts received CYP2D6 inhibitors. Nine pts did not complete the planned 4 months with tamoxifen 20 mg bid. The main reasons were mood disorders, hot flashes, headache and nausea. Self-reported treatment compliance assessed by monthly anonymous questionnaire was ≥ 95%, except 80–95% in 4 pts. Conclusions: This is the first trial reporting the impact of the increase of tamoxifen dose in all CYP2D6 genotypes, including EMs. Dose escalation of tamoxifen increased significantly the plasma level of endoxifen by similar ratio in all genotype subgroups. Because of a huge inter-individual variability genotyping and phenotyping are not adequate surrogate markers of endoxifen level. Very low endoxifen levels are observed even in pts classified as EM. Future trials aiming to improve the plasma level of endoxifen should consider direct measurement of the metabolite in plasma and adjust tamoxifen dose according to the initial level of the metabolite independently of the genotype. Reference 1. A. Dieudonné, Journal of Clinical Oncology, 2011; vol 29, No 15, suppl (May 20, 2011): TPS 140 Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-05.

Effects of cysteine on the pharmacokinetics of tamoxifen in rats with protein-calorie malnutrition

Protein-calorie malnutrition (PCM) could occur frequently in cancer patients and alter the pharmacokinetics of drugs. Also cysteine shows anti-oxidative effect and changes the activities of drug metabolizing enzyme and/or transporters. Herein, we investigated the effects of cysteine on the pharmacokinetics of tamoxifen in rats with protein-calorie malnutrition (PCM).The in vivo pharmacokinetics and in vitro hepatic/intestinal metabolism of tamoxifen were assessed using control, CC (control with cysteine), PCM, PCMC (PCM with cysteine) rats. The effects of cysteine on the intestinal absorption of tamoxifen were further investigated through in vitro transport studies using rat intestine.The AUCs of intravenous tamoxifen in PCM rats were significantly greater than control rats due to the decrease in the hepatic metabolism via CYP3A. In PCMC rats, the elevated AUCs in PCM rats returned to control levels by oral cysteine supplement. The AUC of oral tamoxifen in PCM rats was significantly smaller than in control rats mainly due to the decrease in gastrointestinal absorption. In CC and PCMC rats, oral cysteine supplement enhanced the gastrointestinal absorption of tamoxifen probably via intestinal P-gp inhibition.The present study demonstrated that PCM state and/or oral cysteine supplement had a profound impact on the pharmacokinetics of tamoxifen in rats. If the present rat data are extrapolated to humans, the alterations in tamoxifen absorption and metabolism should be considered in designing a dosage regimen for cancer patients with PCM and/or oral cysteine supplement.

JS4-3 - Recent Progress and Future Perspective of Endocrine Therapy for Breast Cancer

ABSTRACT Endocrine therapy is the classical target therapy applying to major part of breast cancer patients. At present, there are sub-classes of drug called aromatase inhinitor (AI), selective estrogen receptor modulator (SERM), selective estrogen receptor down-regulator (SERD), progestin and estrogen derivatives. Although novel predictive factor for endocrine therapy has never been added to date other than the presence of ER and/or PgR, recent progress gave us some insight of such factors related to patient herself. CYP2D6 genotype for tamoxifen metabolism and body mass index for AI response are those arising from individual patient difference, but related to the response of drug in some degree. In addition, combination with other molecular pathway drugs such as anti-HER2 agents, mTOR inhibitor and anti-angiogenesis drug have been intensively investigated, showing double, triple or more simultaneous pathway blocking approach may have dramatic response to hormone receptor (HR) + breast cancer. From the point of view that these HR + tumors generally showed poor response to conventional chemotherapy, these approaches seem to be leading edge for improvement of this subtype of cancer patients.

Role and pharmacologic significance of cytochrome P‐450 2D6 in oxidative metabolism of toremifene and tamoxifen

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P-450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N-desmethyl (NDM) and 4-hydroxy (4-OH) metabolites during steady-state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4-OH TAM and 4-OH-NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4-OH-NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4-hydroxylation of TOR by CYP2D6. The 4-OH and 4-OH-NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30-fold greater affinity were 35- to 187-fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360-fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4-OH TAM and 4-OH-NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6-inhibiting medications.

295P - Concomitant Use of Antidepressant Drugs and Tamoxifen

ABSTRACT Disclosure All authors have declared no conflicts of interest. The CYP2D6 enzyme is primarily involved in the metabolism of tamoxifen into its most important metabolite endoxifen. The efficacy of tamoxifen therapy may be influenced by genetic polymorphisms of this enzyme and CYP2D6-inhibiting co-medication (e.g. antidepressant drugs). It is therefore generally recommended to avoid potent CYP2D6 inhibitors (e.g. paroxetine, fluoxetine) in tamoxifen patients. In this study, we examined whether women using tamoxifen still received strong CYP2D6-inhibiting antidepressants concomitantly during the period 2005-2010. Drug dispensing data for tamoxifen and seven antidepressants associated with CYP2D6 inhibition (paroxetine, fluoxetine, sertraline, fluvoxamine, (es)citalopram and venlafaxine), were obtained from the community pharmacy database of the PHARMO Institute. This database contains dispensing data of > 3 million people in the Netherlands. Concomitant use of CYP2D6-inhibiting antidepressants in women using tamoxifen, as well as the use of antidepressants in the total population, were determined per year using PASW Statistics 17.0 (SPSS Inc., Chicago, IL). Prevalence of the use of the four most common antidepressants among tamoxifen users, expressed as the number of women receiving an antidepressant concurrently with tamoxifen per 1000 tamoxifen users, is shown in the table. Although there is a downwards trend for paroxetine, this drug is still one of the most frequently used antidepressants in tamoxifen patients. In addition, a similar trend was observed in the total population of the database. Venlafaxine and citalopram, associated with weak CYP2D6-inhibiting properties, are increasingly co-prescribed. 2005 2006 2007 2008 2009 2010 Paroxetine 47.1 44.3 46.6 41.7 36.9 28.8 Fluoxetine 9.5 10.6 9.8 10.7 10.8 9.7 Citalopram 17.0 23.5 22.4 26.2 25.2 28.5 Venlafaxine 18.3 23.8 28.2 27.5 29.1 30.8 Despite the strong biological rationale to avoid potent CYP2D6-inhibiting co-medication in tamoxifen treated patients, paroxetine is still often used along with tamoxifen. In clinical practice, one should be extremely alert to co-medication in these patients. It is advised that strong CYP2D6 inhibitors are switched to weaker CYP2D6-inhibiting alternatives, if possible.

In Silico Modeling of UDP-Glucuronosyltransferase 1A10 Substrates Using the Volsurf Approach

UDP-glucuronosyltransferase 1A10 (UGT1A10) catalyzes glucuronidation of a wide range of chemicals including many drugs. Here, we report the first in silico model quantifying the substrate selectivity and binding affinity (as K(m)) for UGT1A10. The training set for model construction comprises 32 structurally diverse compounds, which are known substrates for UGT1A10. The model was derived by applying the standard VolSurf method involving calculation of VolSurf descriptors and partial least square (PLS) analyses. The yielded PLS model with two components shows statistical significance in both fitting and internal predicting (r(2) = 0.827, q(2) = 0.774). The model predictability was further validated against a test set of 11 external compounds. The activity values for all test substrates were predicted within 1 log unit. Moreover, the model reveals an overlay of chemical features influencing the enzyme-substrate binding. Those include the size and shape, capacity factors, hydrophilic regions, hydrophobic regions, and polarizability. In conclusion, the VolSurf approach is successfully utilized to establish a predictive model for UGT1A10. The derived model should be an efficient tool for high-throughput prediction of UGT1A10 metabolism.

Bioactivation of the Cancer Chemopreventive Agent Tamoxifen to Quinone Methides by Cytochrome P4502B6 and Identification of the Modified Residue on the Apoprotein

The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide ¹⁸⁸FHYQDQE¹⁹⁴ with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.

Common polymorphisms in the estrogen receptor-1 may determine risk of hot flashes in early breast cancer patients using tamoxifen.

526 Background: In breast cancer patients the occurrence of hot flashes as common side effect of tamoxifen therapy may be associated with effective estrogen receptor antagonism dependent on genetic variations of metabolic enzymes and the estrogen receptor. Methods: 742 early breast cancer patients who were randomized to receive tamoxifen, followed by exemestane after 2.5 to 3 years within the Tamoxifen Exemestane Adjuvant Multinational (TEAM) Trial were genotyped for 30 germ line genetic variants of 11 enzymes that are involved in the tamoxifen metabolism and the estrogen receptor 1 (ESR1). These genetic variants were related to the occurrence of hot flashes during the first year of tamoxifen use (primary aim) and during the complete tamoxifen treatment period (secondary aim). A multivariable logistic regression was used to adjust for age and adjuvant chemotherapy. Results: No genetic variant was associated with the occurrence of hot flashes during the first year. Higher age was related to a lower incidence of hot flashes in the first year (adjusted odds ratio 0.94, 95% CI 0.92-0.96; p<0.001). The ESR1 PvuII XbaI CG haplotype (CG/CG vs CG/other + other/other: adjusted odds ratio 0.44, 95% CI 0.21-0.92; p=0.03), ESR1 PvuII XbaI TA haplotype (TA/TA + TA/other vs other/other: adjusted odds ratio 1.86, 95% CI 1.09-3.14; p=0.02) and age (adjusted odds ratio 0.94, 95% CI 0.92-0.97; p<0.001) were associated with the occurrence of hot flashes during the total tamoxifen treatment period. No association was found between the CYP2D6 predicted phenotype and hot flashes. Conclusions: Common polymorphisms in the estrogen receptor-1 might help to predict the occurrence of hot flashes in breast cancer patients treated with adjuvant tamoxifen. If replicated, this may provide clinicians with a tool to offer more personalized hormonal therapy.

Abstract 3781: Pharmacokinetics of endoxifen and tamoxifen in female mice: Implications for comparative in vivo activity studies

Abstract Background: In tamoxifen (TAM) treated patients, both CYP2D6 metabolism and low steady state concentrations of endoxifen (END), the active metabolite of TAM, have been associated with a higher risk of recurrence (Madlensky L, Clin Pharmacol Ther. 2011; 89(5):718-25). END drug development is ongoing at Mayo and NCI, and our previous data demonstrated that 1) END had high oral bioavailability in mice and yielded plasma concentrations and total exposure 20-fold greater as compared to an equivalent oral dose of TAM (MM Ames et al, Cancer Res 2011;70(8 Suppl 1):Abstract nr 3603) and 2) END is superior to TAM in both HER2 (Reinicke AACR 2011) and aromatase expressing (Goetz, SABC 2011) MCF-7 xenograft models. While subcutaneous (SQ) TAM is used by most researchers to study and develop models of TAM resistance, little is known about 1) whether murine and human TAM pharmacokinetics are comparable and 2) whether the route of administration affects concentrations of the active metabolites. Therefore, we conducted murine pharmacokinetic studies evaluating both the oral and SQ administration of TAM and END. Results: Following administration of TAM, concentrations of the active metabolites (END and 4-hydroxy TAM [4HT]) were therapeutic (Cmax- END 35.9 nM, 4HT 62.8 nM) only after a 0.5 mg oral dose of TAM. In contrast, following all doses of SQ TAM, concentrations of the active metabolites (END and 4HT) were sub-therapeutic (Cmax- END 5.2 nM; 4HT 18.7 nM). In contrast to humans, the Cmax of N-desmethyl TAM, the most abundant human TAM metabolite, was < 16.5 nM in mice after oral and SQ TAM. SQ administration of END and TAM resulted in substantially greater bioavailability of the parent drug compared with oral dosing. Following administration of 0.25-5 mg oral END and 0.0625-0.625 mg subcutaneous END, the increase in AUC was greater than dose proportional and active concentrations (>80 nM) were detected in plasma at every dose and both dosing routes. Comparable END and TAM plasma concentrations were achieved with a 0.0625 mg SQ END and a 0.5 mg SQ TAM, respectively. Conclusions: Our data strongly suggest that SQ dosing of TAM results in non-therapeutic concentrations of the active hydroxylated metabolites, and may be a simple pharmacokinetic factor influencing response and resistance in a murine in vivo xenograft model. Given the nonlinear pharmacokinetics and differences in metabolism, the dose and route of END must be carefully selected for comparative studies with TAM. These data support the ongoing human studies designed to bypass the need for TAM metabolism by the direct administration of END. 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 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3781. doi:1538-7445.AM2012-3781

Cancer Patients Have Much to Gain From Psychiatric Treatment

Cancer Patients Have Much to Gain From Psychiatric Treatment

Effect of administration of tamoxifen citrate on xanthine oxidase activity of liver during 3-methylcholanthrene induced carcinogenesis in female albino mice

Tamoxifen citrate, the non-steroidal anti-estrogen and the first selective estrogen receptor modulator is the treatment of choice for patients with all stages of estrogen receptor positive breast cancer. Although Cytochrome P-450s (CYP) are well known to be responsible for much of its metabolism, fragmentary studies have been carried out to analyze the activity of xanthine oxidase, a non-CYP drug metabolizing enzyme, in tamoxifen metabolism. So, the present investigation is aimed to ascertain the effect of tablet and nano-formulation of tamoxifen on xanthine oxidase expression during 3-methylcholanthrene-induced carcinogenesis. It is found that oral administration of tamoxifen in both the formulations significantly (p < 0.01) decreases the xanthine oxidase activity in liver tissue of female mice. We speculate our results to be beneficial for the better understanding of drug metabolism and intensity, which in turn will pave the way for new and efficient nano-formulations of drug coming into therapeutic use.

Physiologically based pharmacokinetic modeling of tamoxifen and its metabolites in women of different CYP2D6 phenotypes provides new insight into the tamoxifen mass balance

Tamoxifen is a first-line endocrine agent in the mechanism-based treatment of estrogen receptor positive (ER+) mammary carcinoma and applied to breast cancer patients all over the world. Endoxifen is a secondary and highly active metabolite of tamoxifen that is formed among others by the polymorphic cytochrome P450 2D6 (CYP2D6). It is widely accepted that CYP2D6 poor metabolizers exert a pronounced decrease in endoxifen steady-state plasma concentrations compared to CYP2D6 extensive metabolizers. Nevertheless, an in-depth understanding of the chain of cause and effect between CYP2D6 genotype, endoxifen steady-state plasma concentration, and subsequent tamoxifen treatment benefit still remains to be evolved. In this study, physiologically based pharmacokinetic (PBPK)-modeling was applied to mechanistically investigate the impact of CYP2D6 phenotype on endoxifen formation in female breast cancer patients undergoing tamoxifen therapy. A PBPK-model of tamoxifen and its pharmacologically important metabolites N-desmethyltamoxifen (NDM-TAM), 4-hydroxytamoxifen (4-OH-TAM), and endoxifen was developed and validated. This model is able to simulate the pharmacokinetics (PK) after single and repeated oral tamoxifen doses in female breast cancer patients in dependence of the CYP2D6 phenotype. A detailed model-based analysis of the mass balance offered support for a recent hypothesis stating a more prominent role for endoxifen formation from 4-OH-TAM. In the future this model provides a good basis to further investigate the linkage of PK, mode of action, and treatment outcome in dependence of factors such as phenotype, ethnicity, or co-treatment with CYP2D6 inhibitors.

The Risk of Recurrence in Breast Cancer Patients Treated with Tamoxifen: Polymorphisms of CYP2D6 and ABCB1

CYP2D6 plays a major role in the metabolism of tamoxifen, and polymorphism of P-glycoprotein has been associated with resistance of many drug therapies. This study investigates the clinical impact of genetic variants of CYP2D6 and ABCB1 in breast cancer patients treated with tamoxifen. Blood samples from 95 breast cancer patients treated with tamoxifen were collected and genotyped for CYP2D6 and ABCB1 variants using allele-specific PCR method. Recurrence risks were calculated using Kaplan-Meier analysis and compared using the log-rank test. Patients carrying CYP2D6*10/*10 and heterozygous null allele (IM) showed higher risks of developing recurrence and metastasis (OR 13.14; 95% CI 1.57-109.94; P = 0.004) than patients with CYP2D6*1/*1 and *1/*10 genotypes. Patients with homozygous CC genotypes of ABCB1 C3435T showed a shorter time to recurrence. Patients who were CYP2D6 IM and homozygous CC genotype of C3435T have statistically significant higher risks of recurrence (P = 0.002). Similarly, median time to recurrence in these patients was only 12 months (95% CI = 0.79-23.2) compared to those without this combination which was 48 months (95% CI = 14.7-81.2). Patients with CYP2D6 IM and homozygous CC genotype of ABCB1 C3435T have shorter times to recurrence. The results confirmed the findings of previous studies and support FDA recommendation to perform pre-genotyping in patients before the choice of therapy is determined in breast cancer patients.

P4-01-16: The Influence of CYP2D6 Genetic Polymorphisms on Variability of Tamoxifen Metabolism in the Lebanese Breast Cancer Population.

Abstract Introduction The selective estrogen receptor modulator tamoxifen is commonly used for the treatment and prevention of breast cancer. Tamoxifen needs to be converted by the highly polymorphic enzyme, CYP2D6, to its active metabolites (4-hydroxytamoxifen and endoxifen) to exert its anti-estrogenic effect. Several clinical trials have shown that CYP2D6 poor metabolizers have decreased benefit from tamoxifen compared to extensive metabolizers. Large ethnic variations have been noticed in the frequency and effect of CYP2D6 polymorphisms. Objectives To assess for the frequency of CYP2D6*3, *4, *5 (nonfunctional alleles with *5 being a gene deletion), *41 (reduced functional allele) as well as CYP2D6*x N (allele copy number) in a sample of Lebanese participants receiving maintenance doses of tamoxifen for early stage breast cancer, and to assess the effect of these polymorphisms on tamoxifen metabolism in the same subjects. Methods 111 (median age= 48.7± 8.6years) Lebanese breast cancer women were recruited. Blood for isolation of DNA and measurement of tamoxifen metabolites was collected. CYP2D6 genotyping was performed using real-time PCR. Plasma levels of tamoxifen were being measured using HPLC. Results The allele frequencies of CYP2D6*3, *4, and *41 were 0.9, 15.9 and 12.1 respectively. 18.9% of patients had more than 2 CYP2D6 copies and 9% had only 1 copy. Tamoxifen plasma concentration didn't significantly vary among the 4 phenotypic groups, although a trend was observed whereby PMs had the highest tamoxifen plasma concentrations and UMs had the lowest tamoxifen plasma concentrations (Tamoxifen median plasma concentrations in μg/L: PM=290, IM=210, EM=190 and UM=170). Furthermore, there was a statistically significant variation of Z-tamoxifen plasma concentrations among the phenotypic groups when PMs and IMs were grouped together: Z-tamoxifen median plasma concentrations in μg/L: PM+IM=130, EM=90 and UM=62 (P=0.03). Conclusion This is the first study in the Middle East that evaluates the frequencies of CYP2D6 polymorhisms in a Lebanese breast cancer population and the effect of these polymorphisms on tamoxifen metabolism. We have shown that CYP2D6 phenotype is significantly correlated with Z-tamoxifen levels. PMs and IM group had the highest Z-tamoxifen plasma concentrations; in contrast UMs had the lowest Z-tamoxifen plasma concentrations. Further recruitment and follow up are being performed to assess for the role of these polymorphisms in disease outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-16.

P2-17-04: Induction of Tamoxifen Metabolism by Rifampicin: A Worrying Drug-Drug Interaction.

Abstract Background Tamoxifen (Tam) undergoes extensive biotransformation into several metabolites, including the highly active metabolite endoxifen. Differences in metabolism, influenced by both genetic and environmental factors, largely contribute to the inter-individual variability in endoxifen plasma concentrations, potentially affecting the efficacy of Tam. Conflicting results between CYP2D6 genotype/phenotype and endoxifen concentrations have been observed. A reason for this discrepancy may be that CYP3A4 may have a more crucial role in the formation of endoxifen than previously thought (de Graan et al, JCO, in press). Hypothesizing that induction of CYP3A4 and CYP2D6 could lead to increased endoxifen levels, a prospective study was activated evaluating the effects of enzyme induction by rifampicin (Rif) on the plasma pharmacokinetics (PK) of Tam and its metabolites. Methods: A randomized cross-over study design was used, with each patient serving as her own control. Breast cancer patients on steady state Tam therapy (20 or 40 mg once daily) were included. Patients underwent two periods of plasma sampling covering 24 hrs each, once using Tam alone, and once after 15 days of oral Rif (600 mg daily) taken in combination with Tam. Patients were randomized for sampling sequence 1) Tam alone followed by Rif/Tam versus 2) Rif/Tam followed by Tam alone. PK sampling in sequence 2 was performed after a 30-day wash-out period of Rif. Tam and its main metabolites ND-Tam, 4-OH-Tam and endoxifen were quantitated by a validated LC-MS/MS method. PK parameters, including area under the plasma-concentration time curve (AUC) and maximum concentration (Cmax) of all four compounds were calculated (WinNonLin program) and compared (with or without Rif) using a two-sided paired t-test. For safety reasons an interim-analysis was performed after 4 patients. Results: The interim-analysis showed that concentrations of Tam and its metabolites were significantly decreased during Rif/Tam co-administration as compared to Tam administration alone (see table). Especially endoxifen exposure was decreased by a mean of 69%. Based on these data it was decided to stop further enrollment in this study. Conclusions: Concentrations of Tam and its main metabolites decreased significantly after induction by rifampicin. Potentially contributing factors include further metabolism of Tam-metabolites into other inactive metabolites or conjugates (i.e. glucuronides) or a decreased oral availability of Tam. Further pharmacokinetic analyses, including the analysis of glucuronides and other metabolites, will be performed to better understand the mechanism behind these findings. Based upon these data, combining rifampicin with Tam should be avoided. Similar drug-drug interactions may exist between Tam and other strong CYP inducers, such as St John's wort and phenytoin. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-17-04.

Assessment of the Impact of CYP3A Polymorphisms on the Formation of α-Hydroxytamoxifen and N-Desmethyltamoxifen in Human Liver Microsomes

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL(int))] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min⁻¹ · pmol cytochrome P450⁻¹). There was no significant difference in CL(int) values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL(int) with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL(int) for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.

SULT1A1 , CYP2C19 and Disease-Free Survival in early Breast Cancer Patients Receiving Tamoxifen

Tamoxifen biotransformation to endoxifen, a potent antiestrogen, is catalyzed by CYP2D6. In addition, CYP2C19 and SULT1A1 have also been implicated in the metabolism of tamoxifen. We sought to evaluate the importance of SULT1A1 copy number and CYP2C19*17 on disease-free survival (DFS) in postmenopausal women randomized to tamoxifen monotherapy in North Central Cancer Treatment Group 89-30-52 from January 1991 to April 1995. We extracted DNA from paraffin-embedded tumors and determined tumor SULT1A1 copy number and CYP2C19*17 genotype. The association of genotype with DFS was determined using the log-rank test. Multivariate cox modeling was performed using traditional prognostic factors, as well as CYP2D6 genotype. SULT1A1 copy number and CYP2C19*17 genotype was determined in 190 out of 256 patients (95% Caucasian). The median follow-up for living patients was 14 years. DFS did not differ according to SULT1A1 copy number (p = 0.482) or CYP2C19*17 genotype (p = 0.667). Neither SULT1A1 copy number or CYP2C19*17 genotype was associated with disease recurrence in this cohort. Future studies are needed to identify whether other genetic and environmental factors which affect tamoxifen metabolism are associated with tamoxifen clinical outcomes.