n-desmethyltamoxifen and Breast-Neoplasms

Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.

Topics: Acid Ceramidase; Antineoplastic Agents, Hormonal; Biotransformation; Breast Neoplasms; Ceramides; Drug Resistance, Neoplasm; Female; Humans; Hydrolysis; Lipid Metabolism; Lysophospholipids; Sphingosine; Tamoxifen

Topics: Antidepressive Agents, Second-Generation; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cyclohexanols; Cytochrome P-450 CYP2D6; Estrogen Receptor Modulators; Estrogen Replacement Therapy; Female; Fluoxetine; Hot Flashes; Humans; Hydroxylation; Paroxetine; Randomized Controlled Trials as Topic; Selective Estrogen Receptor Modulators; Tamoxifen; Venlafaxine Hydrochloride

Over the past decade, the non-steroidal antiestrogen tamoxifen has gained general acceptance for the palliative treatment of breast cancer. Although there has been much interest in the pharmacology of tamoxifen, our knowledge of its metabolism in laboratory animals and patients is incomplete and the precise mechanism of action within target tissue and breast tumor cells is unknown. This review briefly describes the pharmacology of tamoxifen in various laboratory species and patients. Several metabolites of tamoxifen are known and their relative potencies as estrogens and antiestrogens are compared with the parent compound. Apart from monohydroxytamoxifen, none of tamoxifen's metabolites are more potent antiestrogens, but a metabolite in the dog, Metabolite E, is fully estrogenic. Routine assays (tlc, HPLC, glc/ms) are available to detect tamoxifen, N-desmethyltamoxifen, monohydroxytamoxifen, and a newly identified metabolite, designated Metabolite Y, in biological fluids. Continuous therapy with tamoxifen (10 mg bid) produces steady-state levels (100-200 ng/ml serum) within 4 weeks. Levels of N-desmethyltamoxifen are often up to twice the levels achieved with tamoxifen, while levels of monohydroxytamoxifen and Metabolite Y are below 10 ng/ml. Although monohydroxytamoxifen has a high binding affinity for the estrogen receptor, the metabolic activation of tamoxifen is an advantage rather than a requirement for antiestrogenic activity. The action of tamoxifen in vivo is the net result of the individual actions of the parent compound and its metabolites competing for the occupation of receptors within target tissues and tumors.

Topics: Breast Neoplasms; Estrogen Antagonists; Female; Humans; Palliative Care; Receptors, Estrogen; Tamoxifen

Both therapeutic and adverse effects of tamoxifen may be related to its tissue concentrations. We investigated concentrations of tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and N-didesmethyltamoxifen in serum, normal breast, and breast cancer tissues during conventional dosage and two low-dose regimens. Furthermore we studied tamoxifen effects on the cancer proliferation marker Ki-67, and on sex hormone-binding globulin (SHBG).. From September 1999 to August 2001, 120 breast cancer patients were randomized to 20-, 5-, or 1-mg tamoxifen daily. We measured serum and tissue concentrations of tamoxifen and three metabolites after 28 days of treatment, and the changes between baseline and post-treatment levels of SHBG and Ki-67.. The median (range) tamoxifen concentrations (ng/ml) at doses of 1, 5, and 20 mg daily (n = 38, 37, and 36) were 7.5 (2.9-120.9), 25.2 (1.9-180.9), and 83.6 (8.7-134.4) in serum, and 78.2 (35.9-184), 272.3 (122-641), and 744.4 (208.6-2556) in breast cancer tissue, respectively. Tamoxifen levels followed a dose-concentration relationship. The concentrations of tamoxifen and metabolites were related to each other. Serum and tissue concentrations of tamoxifen were associated with corresponding changes of SHBG levels, whereas changes of Ki-67 levels were not related.. Estrogen agonistic effects of tamoxifen on SHBG decreased with lower dosage, whereas tamoxifen effects on Ki-67 expression did not change. This together with a >10-fold variation in serum tamoxifen concentrations and a serum to tissue concentration relationship suggest that tamoxifen treatment may be improved by administration of lower doses and therapeutic drug monitoring.

Topics: Aged; Breast Neoplasms; Chromatography; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Estrogen Antagonists; Female; Humans; Ki-67 Antigen; Middle Aged; Receptors, Estrogen; Sex Hormone-Binding Globulin; Smoking; Tamoxifen; Time Factors

We have shown previously that a reduction from the conventional dose of tamoxifen is associated with a comparable modulation of circulating biomarkers, including insulin-like growth factor-I and cholesterol. In the present study, we have correlated serum tamoxifen elimination with biomarker recovery in healthy subjects completing a 5-year intervention period. Tamoxifen, N-desmethyltamoxifen, and biomarker levels were measured at 0 (baseline), 2, 4, and 6 weeks after completion of treatment in 23 healthy postmenopausal women allocated to tamoxifen 20 mg/day and in 6 women allocated to placebo. Mean (+/-SD) serum tamoxifen and N-desmethyltamoxifen concentrations were, respectively, 141 +/- 50 and 226 +/- 77 ng/ml at baseline, 36 +/- 19 and 99 +/- 46 at 2 weeks, 20 +/- 15 and 61 +/- 37 at 4 weeks, and 12 +/- 9 and 36 +/- 26 at 6 weeks. Serum tamoxifen and N-desmethyltamoxifen half-lives were 9 and 13 days, respectively. Body mass index was associated positively with drug's serum half-life. Compared with baseline values, the percentage increase in total cholesterol, low-density lipoprotein cholesterol, and insulin-like growth factor-I 4 weeks after treatment completion was 5, 9, and 14%, respectively. No change during the 6-week period was observed in the placebo arm. Our findings indicate that the biomarker recovery is slower than serum tamoxifen elimination, suggesting that low tamoxifen concentrations may still exert a biological effect. In addition, the prolonged half-life of tamoxifen and metabolite provides the rationale for a weekly administration of the drug in a preventive context. However, the clinical implications of our findings remain to be defined.

Topics: Aged; Biomarkers; Body Mass Index; Breast Neoplasms; Cholesterol; Cholesterol, LDL; Female; Humans; Insulin-Like Growth Factor I; Middle Aged; Postmenopause; Selective Estrogen Receptor Modulators; Tamoxifen

We studied the bioequivalence of a new once-daily regimen of tamoxifen citrate relative to the standard twice-daily regimen of tamoxifen citrate, an established antiestrogenic treatment for breast cancer.. Of 30 women with breast cancer, 27 completed this open, two-period, crossover randomized trial. During one 3-month period, patients took one standard 10-mg tamoxifen tablet twice daily; during the preceding or following 3-month period, patients took one of the new 20-mg tablets once daily. Pharmacokinetic profiles and safety parameters were assessed at the end of each 3-month treatment period.. Overall, measured concentrations of tamoxifen and its principal active metabolite, N-desmethyltamoxifen, remained relatively constant over the 24-hour sampling periods at the end of each treatment sequence. For both compounds, the percentage differences of the geometric means for all pharmacokinetic parameters indicated bioequivalence of the once-daily regimen of tamoxifen relative to the standard twice-daily regimen. Both treatment sequences were well tolerated; reported adverse events occurred at similar frequencies with the two treatment regimens.. The 20-mg tamoxifen tablet taken once daily was bioequivalent to the 10-mg tamoxifen tablet taken twice daily, with no difference in relative risk. The once-daily treatment is a simpler regimen and may facilitate compliance, which may enhance therapeutic outcomes during long-term treatment of breast cancer.

Topics: Administration, Oral; Aged; Analysis of Variance; Breast Neoplasms; Drug Administration Schedule; Female; Humans; Middle Aged; Tamoxifen; Therapeutic Equivalency

While the presence of oestrogen receptors (ERs) in human breast cancer may determine the biological response to tamoxifen, the extent to which ER status governs tumour tamoxifen accumulation is unclear. We investigated the intra-tumoural disposition of tamoxifen (TAM) and its major metabolite N-desmethyltamoxifen (DMT) in 36 human breast carcinomas following short-term therapy. Steady-state serum concentrations appeared to be reached following 2 weeks therapy, after which no significant difference in the intra-tumoural concentrations of TAM between ER-ve and ER+ve tumours was observed (717.9 +/- 166.4 ng/gm, and 518.6 +/- 109.4 ng/gm, respectively). In patients treated for less than 2 weeks, there was significantly less intra-tumoural TAM in ER-ve compared with ER+ve tumours (120.9 +/- 49.9 ng/gm and 450.1 +/- 75.3 ng/gm, respectively; p < 0.04). The rate of tumour TAM accumulation correlated with duration of therapy only for ER-ve tumours (r = 0.72, p < 0.02), whereas for ER+ve tumours the absolute ER value appeared to be weakly associated with TAM accumulation (r = 0.41; p < 0.05). The intra-tumoural ratio of TAM to DMT reflected the serum concentrations in ER-ve tumours, but in ER+ve tumours relatively more TAM to DMT was observed. A similar intracellular distribution of both TAM and DMT was observed, although following 2 weeks therapy relatively less of each compound was found in the cytosol of ER-ve compared with ER+ve tumours (18% vs 34%). These results demonstrate that ER status may influence the rate of accumulation and intra-cellular distribution of tamoxifen and its metabolites, but not the final concentrations which are achieved. Following steady-state, both ER+ve and ER-ve tumours, not all of which would be expected to respond to the drug, achieve intra-tumoural concentrations 5-7 fold greater than serum. Unlike recent reports on acquired resistance, therefore, de novo resistance to tamoxifen is unlikely to represent an inability of the tumour to achieve adequate intra-tumoural concentrations of the drug or its metabolites.

Topics: Aged; Breast Neoplasms; Drug Administration Schedule; Drug Resistance; Female; Humans; Middle Aged; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Subcellular Fractions; Tamoxifen

The steady-state pharmacokinetics of tamoxifen and its metabolites was studied in sixteen patients with advanced mammary cancer. Patients were randomized to receive tamoxifen given as Tamofen, Leiras, or as Nolvadex, ICI, 20 mg twice daily for 16 weeks in a cross-over study. Plasma and urine samples were analyzed during one dose interval (12 h) after treatment for 8 and 16 weeks. The concentrations of tamoxifen, N-desmethyltamoxifen, N,N-desdimethyltamoxifen, and metabolite Y were determined in plasma and the areas under the plasma level curves were calculated. 4-Hydroxytamoxifen was not found in plasma. In urine samples only tamoxifen and N-desmethyltamoxifen were above the detection limits even though metabolite Y was also analyzed after acid hydrolysis. There were no statistically significant differences in the concentrations of tamoxifen and its metabolites between the two preparations. The results of nonresponders did not differ from those of responders. Liver metastases had no effect on the metabolism of tamoxifen.

Topics: Aged; Breast Neoplasms; Clinical Trials as Topic; Female; Humans; Kinetics; Middle Aged; Neoplasm Metastasis; Random Allocation; Tamoxifen

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytochrome P-450 CYP2D6; Female; Genome-Wide Association Study; Genotype; Humans; Tamoxifen

Tamoxifen, a widely prescribed medication in premenopausal women diagnosed with hormone-dependent breast cancer, is potentially co-prescribed with Hedyotis diffusa (H. diffusa), particularly in Taiwan. However, no related report has investigated the drug-herb interaction of H. diffusa on the pharmacokinetics of tamoxifen and its metabolites. In the present study, male Sprague-Dawley rats were administered different doses of H. diffusa extract for 5 consecutive days prior to the administration of tamoxifen (10 mg/kg). A validated ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system was developed to monitor tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen in rat plasma. Pharmacokinetic results demonstrated that the area under curves (AUCs) of tamoxifen and the relative bioavailability (%) of tamoxifen were dose-dependently decreased (31-68%) by pre-treatment with H. diffusa extract (3 g/kg and 6 g/kg). In addition, the conversion ratio of 4-hydroxytamoxifen was downregulated (0.5-fold change) and the N-desmethyltamoxifen conversion ratio was upregulated (2-fold change) by high-dose H. diffusa extract. As a result, the relative bioavailability and biotransformation changes affect the clinical efficacy of tamoxifen treatment. These preclinical findings reveal a hitherto unreported interaction between tamoxifen and H. diffusa extract that has implications for their therapeutic efficacy in treating breast cancer.

Topics: Animals; Biological Availability; Biotransformation; Breast Neoplasms; Chromatography, Liquid; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Hedyotis; Herb-Drug Interactions; Plant Extracts; Rats; Rats, Sprague-Dawley; Tamoxifen; Tandem Mass Spectrometry

Cannabidiol (CBD) serves as a promising medicine, with few known adverse effects apart from the potential of drug interactions with the cytochrome P450 system. It has been hypothesized drug interactions may occur with chemotherapeutic agents, but no supporting evidence has been published to date.. A 58-year-old female with a history of bilateral breast carcinoma in remission, was treated with tamoxifen for breast cancer prevention for over 6 years. CBD was instituted to treat persistent postsurgical pain, inadequately managed by alternate analgesics. It was postulated that CBD may diminish tamoxifen metabolism by CYP3A4 and 2D6 to form active metabolite endoxifen, which exerts the anticancer benefits. Endoxifen, tamoxifen, N-desmetyltamoxifen and 4-hydroxytamoxifen levels were collected while the patient chronically received CBD 40 mg/day, and after a 60-day washout. Upon discontinuation of CBD 40 mg/day, it was observed that endoxifen levels increased by 18.75% and N-desmethyltamoxifen by 9.24%, while 4-hydroxytamoxifen remained unchanged.. CBD at a low dose of 40 mg/day resulted in the potential inhibition of CYP3A4 and/or CYP2D6. Patients receiving CBD and interacting chemotherapeutic drugs, such as tamoxifen, require monitoring to identify possible subtherapeutic response to treatment. Further pharmacokinetic studies are required to ascertain the dynamics of this drug interaction.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cannabidiol; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Drug Interactions; Female; Humans; Middle Aged; Tamoxifen

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chemotherapy, Adjuvant; Cytochrome P-450 CYP2D6; Dextromethorphan; Female; Genotyping Techniques; Humans; Middle Aged; Tamoxifen

CYP2D6 protein activity can be inferred from the ratio of N-desmethyl-tamoxifen (NDMT) to endoxifen (E). CYP2D6 polymorphisms are common and can affect CYP2D6 protein activity and E level. Some retrospective studies indicate that E < 16 nM may relate to worse outcome.. A target NDMT/E ratio was defined as associated with an E level of 15 nM in the 161 patient Test cohort of tamoxifen-treated patients, dichotomizing them into 'Normal' (NM) and 'Slow' (SM) CYP2D6 metabolizer groups. This ratio was then tested on a validation cohort of 52 patients. Patients were phenotyped based on the standard method (ultrarapid/extensive, intermediate or poor metabolizers; UM/EM, IM, PM) or a simplified system based on whether any variant allele (V) vs wildtype (wt) was present (wt/wt, wt/V, V/V). Comprehensive CYP2D6 genotyping was undertaken on germline DNA.. A target NDMT/E ratio of 35 correlated with the 15 nM E level, dichotomizing patients into NM (<35; N = 117) and SM (>35; N = 44) groups. The ratio was independently validated by a validation cohort. The simplified system was better in predicting patients without slow metabolism, with specificity and sensitivity of 96% and 44% respectively, compared with the standard method - sensitivity 81% and specificity 83%.. The simplified classification system based on whether any variant was present better identified patients who were truly not CYP2D6 slow metabolizers more accurately than the current system. However, as CYP2D6 genotype is not the only determinant of endoxifen level, we recommend that direct measurement of endoxifen should also be considered.

Topics: Adult; Aged; Alleles; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytochrome P-450 CYP2D6; Drug Monitoring; Female; Genotype; Humans; Middle Aged; Phenotype; Polymorphism, Single Nucleotide; Prospective Studies; Retrospective Studies; Tamoxifen

Vaginally delivered tamoxifen is being developed as alternative to estrogen-based therapies for the treatment of vulvar and vaginal atrophy (VVA) symptoms in subjects at high risk for breast cancer, undergoing treatment for breast cancer with aromatase inhibitors or are breast cancer survivors. Tamoxifen (1 or 20 mg) was administered intra-vaginally to female rabbits once-daily over a 28-day period to assess its pharmacokinetics, systemic exposure and local vaginal tolerance. Plasma samples were taken to assess concentrations of tamoxifen and its metabolites 4-hydroxytamoxifen and N-desmethyltamoxifen over the first day of vaginal administration and following the last dose on Day 28. In-life observations included evaluation of the vaginal region for signs of irritation. At necropsy, vaginal irritation was assessed by the method of Eckstein which reflect collective histopathological grading of four parameters within the vagina including epithelial morphology, leukocytic infiltration, congestion, and edema. Uterine effects of vaginal tamoxifen were also assessed. Plasma concentrations of tamoxifen were higher following administration of 20 mg tamoxifen compared to 1 mg tamoxifen at both Day 1 and Day 28. The metabolite 4-hydroxytamoxifen could not be detected on Day 1 and concentrations were low at Day 28 at the 1 mg tamoxifen dose. 4-Hydroxytamoxifen concentrations were low, but detectable at Day 1 and 28 following administration of 20 mg tamoxifen. The metabolite N-desmethyltamoxifen was undetectable at the 1 mg and 20 mg doses on Day 1; it remained undetectable at the 1 mg tamoxifen dose at Day 28. N-desmethyltamoxifen was detected over the first 8 h of Day 28 then fell below the quantitation limits. There was little to no vaginal or systemic accumulation of tamoxifen following once-daily dosing for 28 days. Tamoxifen accounted for more than 85% of the total systemic exposure compared to its metabolites, 4-hydroxytamoxifen, and N-desmethyltamoxifen. There was essentially no detectable vaginal irritation evident over the course of the study. At necropsy the individual Eckstein scores (maximum score of 16) of the proximal, mid, and distal vagina of females in the 1 mg and 20 mg dose groups were generally comparable in both groups and ranged from minimal to mild magnitude (1 mg dose group: ranging from 1 to 3 in the proximal vagina, 4 to 5 in the mid vagina, and 3 to 7 in the distal vagina; 20 mg dose group: ranging from 3 to 5 in the proximal vagina, 4 to

Topics: Administration, Intravaginal; Animals; Atrophy; Breast Neoplasms; Female; Rabbits; Tamoxifen; Vagina

Tamoxifen is considered a prodrug of its active metabolite endoxifen, which is dependent on the CYP2D6 and CYP3A enzymes. Tamoxifen pharmacokinetic variability influences endoxifen exposure and, consequently, its clinical outcome. This study investigated the impact of hormonal status on the pharmacokinetics of tamoxifen and its metabolites in TAM-treated breast cancer patients.. TAM-treated breast cancer patients (n = 40) previously believed to have CYP3A activity within the normal range based on oral midazolam and phenotyped as CYP2D6 normal metabolizers using oral metoprolol were divided into two groups according to premenopausal (n = 20; aged 35-50 years) or postmenopausal (n = 20; aged 60-79 years) status. All patients were treated with 20 mg/day tamoxifen for at least three months. Serial plasma samples were collected within the 24 h dose interval for analysis of unchanged tamoxifen, endoxifen, 4-hydroxytamoxifen and N-desmethyltamoxifen quantified by LC-MS/MS. CYP activities were assessed using midazolam apparent clearance (CYP3A) and the metoprolol/alfa-hydroxymetoprolol plasma metabolic ratio (CYP2D6). CYP3A4, CYP3A5 and CYP2D6 SNPs and copy number variation were investigated using TaqMan assays.. Postmenopausal status increased steady-state plasma concentrations (Css) of tamoxifen (116.95 vs 201.23 ng/mL), endoxifen (8.01 vs 18.87 ng/mL), N-desmethyltamoxifen (485.16 vs 843.88 ng/mL) and 4-hydroxytamoxifen (2.67 vs 4.11 ng/mL). The final regression models included hormonal status as the only predictor for Css of tamoxifen [β-coef ± SE, p-value (75.03 ± 17.71, p = 0.0001)] and 4-hydroxytamoxifen (1.7822 ± 0.4385, p = 0.0002), while endoxifen Css included hormonal status (8.578 ± 3.402, p = 0.02) and race (11.945 ± 2.836, p = 0.007). For N-desmethyltamoxifen Css, the final model was correlated with hormonal status (286.259 ± 76.766, p = 0.0007) and weight (- 8.585 ± 3.060, p = 0.008).. The premenopausal status was associated with decreased endoxifen plasma concentrations by 135% compared to postmenopausal status. Thus, the endoxifen plasma concentrations should be monitored mainly in the premenopausal period to maintain plasma levels above the efficacy threshold value.. RBR-7tqc7k.

Topics: Adult; Aged; Breast Neoplasms; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Female; Humans; Middle Aged; Polymorphism, Single Nucleotide; Postmenopause; Premenopause; Tamoxifen

Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer.. We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured.. The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/mL (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/mL (10.4 ± 5.2, mean ± SD), that is, 6-fold variation for both.. Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

Topics: Aged; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chromatography, High Pressure Liquid; Estradiol; Female; Follicle Stimulating Hormone; Humans; Luteinizing Hormone; Tamoxifen; Tandem Mass Spectrometry

A method was developed for the analysis of tamoxifen and its main derivatives (4-hydroxytamoxifen, N-desmethyl-tamoxifen, tamoxifen-N-oxide and endoxifen) in human plasma, using micellar liquid chromatography coupled with fluorescence detection. Analytes were off-line derivatized by sample UV-irradiation for 20 min to form the photocycled fluorescent derivatives. Then samples were diluted, filtered and directly injected, thus avoiding extraction steps. The analytes were resolved using a mobile phase containing 0.08 M SDS-4.5% butanol at pH 3 running at 1.5 mL/min through a C18 column at 40°C, without interferences from endogenous compounds in plasma. Excitation and emission wavelengths were 260 and 380 nm, respectively. The chromatographic analysis time was less than 40 min. The analytical methodology was validated following the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of: selectivity, linear range (0.3-15 μg/mL), linearity (r(2)>0.999), sensitivity (LOD, 65-80 ng/mL; LOQ, 165-200 ng/mL), intra- and interday accuracy (-12.2-11.5%) and precision (<9.2%) and robustness (<6.3%). The method was used to quantify the tamoxifen and tamoxifen derivatives in several breast cancer patients from a local hospital, in order to study the correlation between the genotype of the patient and the ability to metabolize tamoxifen.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Chromatography, Liquid; Female; Fluorescence; Humans; Limit of Detection; Micelles; Tamoxifen

A LC-MSMS method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots samples was developed and validated. The method employs an ultrasound-assisted liquid extraction and a reversed phase separation in an Acquity(®) C18 column (150×2.1 mm, 1.7 µm). Mobile phase was a mixture of formic acid 0.1% (v/v) pH 2.7 and acetonitrile (gradient from 60:40 to 50:50, v/v). Total analytical run time was 8 min. Precision assays showed CV % lower than 10.75% and accuracy in the range 94.5 to 110.3%. Mean analytes recoveries from DBS ranged from 40% to 92%. The method was successfully applied to 91 paired clinical DBS and plasma samples. Dried blood spots concentrations were highly correlated to plasma, with rs>0.83 (P<0.01). Median estimated plasma concentrations after hematocrit and partition factor adjustment were: TAM 123.3 ng mL(-1); NDT 267.9 ng mL(-1), EDF 10.0 ng mL(-1) and HTF 1.3 ng mL(-1,) representing in average 98 to 104% of the actually measured concentrations. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold related to better prognosis (5.9 ng mL(-1)). The procedure has adequate analytical performance and can be an efficient tool to optimize adjuvant breast cancer treatment, especially in resource limited settings.

Topics: Adult; Aged; Antineoplastic Agents, Hormonal; Biotransformation; Breast Neoplasms; Chromatography, High Pressure Liquid; Dried Blood Spot Testing; Drug Monitoring; Female; Humans; Limit of Detection; Liquid-Liquid Extraction; Middle Aged; Sonication; Tamoxifen; Tandem Mass Spectrometry

In view of the large variability on therapeutic response and the multiple factors associated to tamoxifen (TAM) metabolic activation, this study aimed to evaluate the effect of CYP2D6 and CYP3A4 phenotypes, drug interactions, and vitamin D exposure on TAM metabolism in a group of breast cancer patients.. Trough blood samples were collected from 116 patients. TAM and metabolites endoxifen (EDF), N-desmethyltamoxifen, and 4-hydroxytamoxifen (HTF) were measured in plasma by liquid chromatography-tandem mass spectrometry. CYP2D6 and CYP3A4 phenotyping were obtained according to [dextromethorphan]/[dextrorphan] and [omeprazole]/[omeprazole sulfone] metabolic ratios, measured by high-performance liquid chromatography in plasma collected 3 hours after oral administration of 33 mg of dextromethorphan and 20 mg of omeprazole. Vitamin D3 was measured in plasma by high-performance liquid chromatography-ultraviolet. Data on concomitant use of drug considered as CYP2D6 and CYP3A4 inhibitor or inducer and vitamin D supplementation were recorded.. About 20% of patients had reduced CYP2D6 metabolic activity and 7% CYP3A4 impaired metabolism. EDF levels diminished proportionally to the reduction of CYP2D6 metabolic activity (poor metabolizer 2.79 ng·mL, intermediate metabolizer (IM) 5.36 ng·mL, and extensive metabolizer 10.65 ng·mL, P < 0.01). Median plasma levels of TAM (161.50 ng·mL) and HTF (1.32 ng·mL) in CYP2D6 IM/CYP3A4 poor metabolizer patients were higher (P < 0.05) than those from CYP2D6 IM/CYP3A4 extensive metabolizer patients (122.07 ng·mL and 0.61 ng·mL, respectively). Seasons contributed to the interpatient variability of EDF and HTF levels; summer concentrations were 24% and 42% higher compared with winter. Vitamin D3 was not associated to CYP3A4 metabolic activity, indicating that other mechanisms might be involved in the relation between TAM metabolism and vitamin D exposure.. CYP3A4 contributes to the bioactivation of TAM through formation of HTF and becomes increasingly important in case of reduced or absent CYP2D6 activity. EDF and HTF exposure were associated to seasonal variations, with considerable higher plasma concentrations during summer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Drug Interactions; Female; Humans; Middle Aged; Phenotype; Tamoxifen; Tandem Mass Spectrometry; Vitamin D

Tamoxifen is a prodrug that is metabolically activated by 4-hydroxylation to the potent primary metabolite 4-hydroxytamoxifen (4OHT) or via another primary metabolite N-desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti-oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment).. The biological effects of tamoxifen and its metabolites on cell growth and oestrogen-responsive gene modulation were evaluated in a panel of oestrogen receptor-positive breast cancer cell lines. Actual clinical levels of tamoxifen metabolites in breast cancer patients were used in vitro along with actual levels of oestrogens observed in premenopausal patients taking tamoxifen.. Tamoxifen and its primary metabolites (4OHT and NDMTAM) only partially inhibited the stimulant effects of oestrogen on cells. The addition of endoxifen at concentrations corresponding to different CYP2D6 genotypes was found to enhance the anti-oestrogenic effect of tamoxifen and its metabolites with an efficacy that correlated with the concentration of endoxifen; at concentrations corresponding to the extensive metabolizer genotype it further inhibited the actions of oestrogen. In contrast, lower concentrations of endoxifen (intermediate and poor metabolizers) had little or no anti-oestrogenic effects.. Endoxifen may be a clinically relevant metabolite in premenopausal patients as it provides additional anti-oestrogenic actions during tamoxifen treatment.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP2D6; Female; Genetic Variation; Genotype; Humans; In Vitro Techniques; MCF-7 Cells; Premenopause; Tamoxifen

A selective and accurate analytical method is needed to quantify tamoxifen and its phase I metabolites in a prospective clinical protocol, for evaluation of pharmacokinetic parameters of tamoxifen and its metabolites in adjuvant treatment of breast cancer. The selectivity of the analytical method is a fundamental criteria to allow the quantification of the main active metabolites (Z)-isomers from (Z)'-isomers. An UPLC-MS/MS method was developed and validated for the quantification of (Z)-tamoxifen, (Z)-endoxifen, (E)-endoxifen, Z'-endoxifen, (Z)'-endoxifen, (Z)-4-hydroxytamoxifen, (Z)-4'-hydroxytamoxifen, N-desmethyl tamoxifen, and tamoxifen-N-oxide. The validation range was set between 0.5ng/mL and 125ng/mL for 4-hydroxytamoxifen and endoxifen isomers, and between 12.5ng/mL and 300ng/mL for tamoxifen, tamoxifen N-desmethyl and tamoxifen-N-oxide. The application to patient plasma samples was performed.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Calibration; Chromatography, Liquid; Drug Monitoring; Female; France; Humans; Metabolic Detoxication, Phase I; Reference Standards; Registries; Reproducibility of Results; Selective Estrogen Receptor Modulators; Spectrometry, Mass, Electrospray Ionization; Tamoxifen; Tandem Mass Spectrometry

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

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytochrome P-450 CYP2D6; Female; Humans; Middle Aged; Tamoxifen

An association between CYP2D6 variation and clinical outcomes among women with breast cancer treated with tamoxifen (TAM) has been demonstrated, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes. This association is mainly due to the CYP2D6-mediated hydroxylation of N-desmethyltamoxifen (NDT) to yield endoxifen (EDF), which because of its high antiestrogenic potency, is mainly responsible for the therapeutic efficacy of TAM. The aim of this study was to evaluate the relation of CYP2D6 genotyping and phenotyping with EDF levels and [NDT]/[EDF] metabolic ratio in breast cancer patients from South of Brazil under TAM therapy.. Trough blood samples were collected from 97 patients. CYP2D6 genotyping was performed with a luminex assay and calculation of genotypic activity scores. Tamoxifen and metabolites EDF, NDT, and 4-hydroxy-TAM were measured in plasma by high performance liquid chromatography with photo diode array detector. CYP2D6 phenotyping was performed by the determination of dextromethorphan (DMT) and dextrorphan (DTF) by high-performance liquid chromatography with fluorescence detection at plasma collected 3 hours after oral administration of 33 mg of DMF. Phenotypes were given according to [DMT]/[DTF] metabolic ratio.. CYP2D6 genotyping indicated a prevalence of 4.1% poor metabolizer, 4.1% intermediate metabolizer, 49.5% extensive metabolizer slow activity, 39.2% extensive metabolizer fast activity, and 3.1% ultrarapid metabolizer. Genotype (genotypic activity scores) was significantly correlated with phenotype ([DMT]/[DTF]), with a moderate association (rs = -0.463; P < 0.001). Median plasma concentrations (nanograms per milliliter; N = 97) were TAM 57.17; 4-hydroxy-TAM 1.01; EDF 6.21; NDT 125.50. EDF levels were lower in poor metabolizers than that in extensive metabolizers (P < 0.05). Phenotype showed stronger, but still moderate, association with EDF and [NDT]/[EDF] than genotype (r = -0.507, r = 0.625, P < 0.001 versus r = 0.356, r = 0.516, P < 0.01). Phenotype accounted for 26% of the variability in EDF levels and 38% of [NDT]/[EDF], whereas genotype accounted for 12% and 27%, respectively.. CYP2D6 genotyping and/or phenotyping could not fully predict EDF concentrations. Monitoring EDF itself could be considered during TAM therapy.

Topics: Adult; Aged, 80 and over; Brazil; Breast Neoplasms; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2D6 Inhibitors; Dextromethorphan; Dextrorphan; Female; Genotype; Humans; Hydroxylation; Middle Aged; Phenotype; Tamoxifen

Endocrine therapy for hormone-sensitive breast cancer is a well-established treatment option, both in adjuvant and palliative settings. For patients undergoing chronic hemodialysis, only scant pharmacokinetic data have been published for tamoxifen, and no data have been published for anastrozole. We therefore measured plasma levels of tamoxifen, its major metabolite, N-desmethyl tamoxifen, and anastrozole in a breast cancer patient undergoing chronic hemodialysis. Clinical tolerability was good. The blood levels for tamoxifen, N-desmethyl tamoxifen and anastrozole were within the expected therapeutic ranges. From this study, we can conclude that endocrine therapy for breast cancer with tamoxifen or anastrozole seems feasible and safe for patients undergoing chronic hemodialysis.

Topics: Anastrozole; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Female; Humans; Middle Aged; Neoplasm Metastasis; Nitriles; Renal Dialysis; Tamoxifen; Triazoles

We have developed a method for the determination of tamoxifen (tam) and its metabolites 4-hydroxytamoxifen (4OHtam), N-demethyltamoxifen (NDtam), N-dedimethyltamoxifen (NDDtam), tamoxifen-N-oxide (tamNox), and 4-hydroxy-N-demethyltamoxifen (4OHNDtam) in 50 microl human serum. Serum proteins were precipitated with acetonitrile. Deuterated-tamoxifen (D5 tam) was added as internal standard. Sample supernatant was injected into an on-line reversed-phase extraction column coupled with a C18 analytical column and analytes were detected by tandem mass spectrometry. The lower limits of quantification were 0.25 ng/mL for 4OHtam, NDtam and tam, 1.0 ng/mL for NDDtam and tamNox. Ranges of within- and between-day variation were 2.9-15.4% and 4.4-12.9%, respectively.

Topics: Breast Neoplasms; Chemical Fractionation; Chromatography, Liquid; Humans; Mass Spectrometry; Sensitivity and Specificity; Tamoxifen

The positive effects of tamoxifen (TAM) on breast cancer recurrence and survival as well as on overall mortality have led to its use as the predominant adjuvant therapy among women with breast cancer. However, the association of TAM intake with undesirable side effects has been reported in numerous studies. This analysis was carried out to assess whether the concentrations of TAM or TAM metabolites, N -desmethyltamoxifen ( N -DMT) and 4-hydroxytamoxifen (4-OHT), were associated with self-reported side effects of TAM. Participants were 99 breast cancer patients who had been taking TAM for at least 30 days. Each participant completed a questionnaire that was used to ascertain whether she experienced certain specific symptoms while taking TAM. In addition, each woman provided a blood sample that was used to measure plasma concentrations of TAM, N -DMT, and 4-OHT by high performance liquid chromatography. Results of the analysis showed that women who experienced at least one TAM-related side effect had significantly higher levels of TAM than women not experiencing any TAM-related side effects. Furthermore, women who reported experiencing visual problems had significantly higher levels of both TAM and N -DMT compared to those women who reported experiencing no visual problems. The levels of 4-OHT were negatively associated with the occurrence of vaginal discharge. The results of this study suggest that the self-reported occurrence of certain symptoms during TAM treatment is related to TAM metabolism. Future studies should assess subgroups of women with specific TAM and TAM metabolite profiles to determine whether alternate, equally effective therapies would decrease their risk of experiencing certain undesirable side effects.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Female; Hot Flashes; Humans; Mental Disorders; Middle Aged; Nausea; Nervous System Diseases; Surveys and Questionnaires; Tamoxifen; Vaginal Diseases

Although tamoxifen (TAM) is the predominant adjuvant therapy for estrogen receptor positive (ER(+)) breast tumors, 50% of breast cancer patients do not respond positively to this therapy, or they experience adverse side effects. This variability in TAM responsiveness may be due to differences in TAM metabolism that stem from differences in race, age, and body mass index (BMI). Thus, the purpose of this study was to test the hypothesis that race, age, and BMI are associated with the metabolism of TAM to two primary metabolites, N-desmethyltamoxifen (N-DMT) and 4-hydroxytamoxifen (4-OHT).. The study design was cross-sectional, and data were analyzed using independent sample t tests and multiple linear regression models. Breast cancer patients (n = 99) taking TAM for at least 30 days were recruited from a local hospital clinic. Each participant provided informed consent, completed a questionnaire, and donated a blood sample. The questionnaire was used to ascertain race, age, and BMI. The blood samples were used to measure plasma concentrations of TAM, N-DMT, and 4-OHT.. Plasma concentrations of TAM, N-DMT, and 4-OHT differed among individual patients. Age, but not race and BMI, was positively associated with plasma concentrations of TAM and N-DMT, even after adjustment for potential confounders (p = 0.02 for TAM and p = 0.03 for N-DMT).. This study suggests that aging may alter the metabolism of TAM. As increased levels of TAM and TAM metabolites may provide a possible explanation for why older women taking TAM are at increased risk for adverse side effects, future studies should determine whether age-related differences in the concentrations of TAM and TAM metabolites are associated with differences in TAM toxicity or responsiveness.

Topics: Adult; Aged; Aging; Antineoplastic Agents, Hormonal; Body Mass Index; Breast Neoplasms; Cross-Sectional Studies; Ethnicity; Female; Humans; Hydroxytestosterones; Maryland; Middle Aged; Neoplasms, Hormone-Dependent; Surveys and Questionnaires; Tamoxifen

The antiestrogen tamoxifen (TAM) is extensively metabolized by cytochrome P-450 in humans and rodents. The active, estrogen receptor-binding metabolites, 4-hydroxy TAM (OHT) and N-desmethyl TAM (DMT) have been well characterized. We showed that the s.c. injection of 1 mg/kg TAM in adult female Sprague Dawley rats bearing carcinogen-induced mammary tumors resulted in rapid serum decline of parent TAM but higher exposure of the metabolites, OHT and DMT. We found for the first time that the administration of TAM for a short time resulted in a delayed induction of caspase activity and apoptosis within the mammary tumors. When TAM, OHT, or DMT was added to human breast cancer cell lines in culture, each elicited a time- and dose-dependent induction of caspase activity, preceding apoptosis. Importantly, pretreatment of the cells with a pharmacological inhibitor of caspases [benzyloxy Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)] blocked apoptosis induced by all three of the compounds, implicating a critical role of caspases in TAM-, OHT-, or DMT-induced apoptosis. The results obtained from these studies suggest that one possible mechanism of inhibition of mammary carcinogenesis and tumor growth in vivo may be the induction of caspase-dependent apoptosis, and that the metabolites OHT and DMT may contribute to the antitumor effect of TAM.

Topics: Animals; Apoptosis; Biotransformation; Breast Neoplasms; Caspases; Enzyme Activation; Enzyme Induction; Estrogen Receptor Modulators; Female; Humans; Mammary Neoplasms, Experimental; Rats; Rats, Sprague-Dawley; Tamoxifen; Tumor Cells, Cultured

A sensitive (200 ng/g) and selective reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen, 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT) in tumour tissue taken from patients undergoing tamoxifen therapy. A muBondapak C18 10 microm column (30 cm x 3.8 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 9 (89:11, v/v). Sample preparation was carried out using a C2 (500 mg sorbent, 3 ml reservoirs) solid-phase extraction method, and extraction efficiencies were followed in individual extracts using a [3H]TAM radiolabelled spike (10000 dpm), with a range of 60-90%. Accuracy and precision (standard deviation) as determined from tumour spiked with radioinert tamoxifen and its metabolites ranged from 83.4-92.3% (+/-23-33%) at 20 microg/g; 85.2-87.7% (+/-18-23%) at 2 microg/g; 88-101% (+/-15-50%) at 0.2 microg/g and 63-94% (+/-13-24%) at 0.02 microg/g. Results from seventy-two patients show mean values (+/-S.D.) of 174+/-203 ng/g for 4-OH; 783+/-1326 ng/g for DMT and 410+/-458 ng/g for TAM, variations reflecting heterogeneity in levels between patients. This methodology can be routinely applied to the determination of tamoxifen and its metabolites in tumour tissues from patients undergoing tamoxifen therapy.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Chromatography, High Pressure Liquid; Female; Humans; Reproducibility of Results; Tamoxifen

Tamoxifen is an antiestrogen used in adjuvant therapy of breast carcinoma and could potentially prevent the development of mammary cancer. While it is widely clinically used, its exact mechanisms of action are not yet fully elucidated. MCF-7/6 cells are estrogen receptor-positive invasive human breast cancer cells with a functionally inactive cell surface E-cadherin. In this study, we report that tamoxifen, and to a lesser extent its metabolites 4-OH-tamoxifen and N-desmethyltamoxifen, restore the function of E-cadherin in MCF-7/6 cells. In an aggregation assay, 10(-6) M tamoxifen significantly increases the aggregation of MCF-7/6 cells. This effect is abrogated by a monoclonal antibody against E-cadherin (HECD-1), is fast (within 30 min), and does not require de novo protein synthesis. Tamoxifen was also found to inhibit the invasion of MCF-7/6 cells in organ culture. Our data is the first demonstration that tamoxifen can activate the function of an invasion suppressor molecule and suggest that the restoration of E-cadherin function may contribute to the therapeutic benefit of tamoxifen in breast cancer patients.

Topics: Breast Neoplasms; Cadherins; Calcium; Cell Adhesion; Female; Humans; Insulin-Like Growth Factor I; Neoplasm Invasiveness; Phenotype; Tamoxifen; Tumor Cells, Cultured

The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines. The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells. Modification was observed at clinically achievable anti-oestrogen concentrations. Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.

Topics: Animals; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carrier Proteins; Cell Division; CHO Cells; Cricetinae; Doxorubicin; Drug Interactions; Drug Resistance; Drug Screening Assays, Antitumor; Epitopes; Estrogen Antagonists; Humans; Lung Neoplasms; Membrane Glycoproteins; Tamoxifen; Toremifene; Tumor Cells, Cultured

To better understand the mechanism of action of antiestrogens, the growth-inhibitory effect of tamoxifen and its main metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, was studied in 6 breast cancer cell lines characterized by different steroid receptor contents. On the basis of the results, our cell lines could be classified into three groups: a first group, including 734B and ZR-75.1 cell lines, characterized by a clear endocrine-dependent behavior, in which cells were sensitive to antiestrogens although to different degrees; a second group, including MDA-MB 231 and BT20 cell lines, characterized by a clear endocrine-insensitive behavior, in which cells were affected only by the highest (10(-6) M) antiestrogen concentration; a third group, including MCF7 and T47D cell lines, characterized by a peculiar behavior. The T47D cell line displayed an increased growth rate after treatment with all three antiestrogens considered. Despite the positive receptor content in the MCF7 cell line, only 4-hydroxytamoxifen showed a clear antiestrogen dose-dependent effect, whereas tamoxifen decreased the cell growth rate only at lower concentrations (10(-8) and 10(-7) M). These results and the well-known heterogeneity of human breast tumors explain the failure of antiestrogen treatment in a certain percentage of patients with breast cancer with a positive estrogen receptor status.

Topics: Breast Neoplasms; Cell Division; DNA, Neoplasm; Estradiol; Estrogen Antagonists; Estrogens; Humans; Kinetics; Neoplasms, Hormone-Dependent; Tamoxifen; Tumor Cells, Cultured

Serum concentrations of tamoxifen, 4-OH-tamoxifen, N-desmethyltamoxifen, and metabolites E and Y were assayed to assess the variation of tamoxifen-metabolism during short-term and long-term endocrine treatment for breast cancer. Once steady-state was achieved, serum levels of tamoxifen and its metabolites in individual patients were stable in the short (10 weeks) and long term (over 7 years) (coefficient of variation [CV], 10-15%), but the variation between individuals (CV 50-70%) was high. Serum tamoxifen and N-desmethyltamoxifen levels were not correlated with indices of obesity. Thus this does not explain the large variation between individuals. In addition to the metabolites that were measured, 4-hydroxy-N-desmethyltamoxifen was tentatively identified in patients' serum. Overall, this study demonstrated that the metabolites of tamoxifen are stable (i.e. no metabolic tolerance) for up to 10 years of drug administration.

Topics: Breast Neoplasms; Drug Stability; Estrogen Antagonists; Humans; Tamoxifen; Time Factors

The effects of tamoxifen, three of its in vivo metabolites and 3-hydroxytamoxifen on cellular proliferation and the induction of four oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-25 and cathepsin D) have been measured in MCF-7 breast cancer cells in phenol red-free culture medium. Tamoxifen and 3-hydroxytamoxifen acted as partial oestrogens to stimulate cell growth and the levels of the pNR-2 and pNR-25 RNAs. They were full oestrogens for the induction of cathepsin D RNA and induced the pNR-1 RNA above the level found in oestrogen-treated cells. N-Desmethyltamoxifen and 4-hydroxytamoxifen behaved like tamoxifen except that N-desmethyltamoxifen did not induce the pNR-2 RNA and was only a partial oestrogen for the induction of cathepsin D RNA, and 4-hydroxytamoxifen did not induce the pNR-2 or pNR-25 RNAs. In the presence of oestradiol, the four anti-oestrogens prevented the stimulation of growth and reduced (pNR-2 and pNR-25) or increased (pNR-1) the RNA levels to those present in MCF-7 cells treated with the anti-oestrogen alone. In contrast, for cathepsin D RNA levels there was a synergistic effect of the anti-oestrogens and oestradiol. The concentration at which each anti-oestrogen was effective was related to its affinity for the oestrogen receptor. Metabolite E was a full oestrogen for the induction of cell proliferation and the oestrogen-regulated RNAs. pNR-25 and pNR-2 RNA levels correlated most closely with effects on cell proliferation. These RNAs are therefore potentially the most useful for predicting the response of breast cancer patients to tamoxifen therapy.

Topics: Breast Neoplasms; Cell Division; Estradiol; Estrogen Antagonists; Gene Expression Regulation; Humans; RNA, Neoplasm; Tamoxifen; Tumor Cells, Cultured

The qualitative and quantitative importance of tamoxifen (TMX) metabolism in vivo led us to investigate further the metabolic profile of this major anti-oestrogenic drug in a significant group of 81 breast cancer patients and to evaluate the respective in vitro activity of each metabolite. TMX and its four metabolites described until now (NDT, 4-OHT, Y, Z) were measured in blood (HPLC method) at the time of first drug intake and at the steady state. Between these two states, the unchanged drug relative proportion dropped from 65% to 27%. Demethylation was the major metabolic pathway. For 13 clinically evaluable patients, there was no significant difference in the distribution of serum levels of TMX and metabolites as a function of response to treatment. In vitro studies were performed on two human breast cancer cell lines: MCF-7, oestrogen receptor and progesterone receptor positive (ER+, PR+) and CAL-18 B (ER-, PR-). Cytostatic effects were evaluated by the tritiated thymidine incorporation test. TMX and all metabolites were active on these two cell lines, but the 50% inhibitory concentrations (IC50) were 4-250-fold higher in CAL-18 B than in MCF-7, depending on the metabolite considered. For the MCF-7 cells only, the antiproliferating activity was parallel to the relative binding affinity for ER. Moreover, for the MCF-7 cells only, the effects of these drugs were partially reversed by oestradiol (E2), the higher the metabolite affinity for ER, the lower the reversal efficacy. These compounds were tested in mixtures at proportions duplicating those found in patients after initial drug intake (mixture D1), and the steady state (mixture Css). The mixtures were also compared to the equimolar unchanged drug. No differences were seen among these three experimental conditions for either MCF-7 or CAL-18 B. A dose-effect relationship was noted. Overall, TMX and its metabolites exert a dual effect: when concentrations are below a threshold between 2 x 10(-6) and 10(-5) M, the drugs are mainly cytostatic; this effect is related to their affinity for ER. At higher relevant clinical concentrations, a cytotoxic activity is observed and it appears independent of the presence of ER.

Topics: Aged; Breast Neoplasms; Estrogen Antagonists; Humans; Tamoxifen; Tumor Cells, Cultured

The bioavailability of two tamoxifen preparations (Tamoplex and Nolvadex) was compared in a multiple dose two-way cross-over design in twelve breast cancer patients. The formulations were found to be bioequivalent. Mean steady-state serum levels of tamoxifen and N-desmethyltamoxifen were 133 ng/ml and 242 ng/ml, respectively, at a single daily dose of three tablets of Tamoplex 10 mg and 128 ng/ml and 248 ng/ml, respectively, at a single daily dose of three tablets of Nolvadex 10 mg. While there was a large interpatient as well as intrapatient variability in steady-state levels of tamoxifen and N-desmethyltamoxifen, their ratio appeared to be constant in the individual patient.

Topics: Adult; Aged; Biological Availability; Breast Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Middle Aged; Tamoxifen

Recent efforts to improve response rates in advanced breast cancer have used short, alternating courses of antiestrogen therapy followed by estrogen priming to cytokinetically enhance tumor cell sensitivity to antimetabolites. Based on recent in vitro and in vivo studies, we have introduced a chemoendocrine regimen that uses prolonged courses of estrogen priming. The present protocol consists of alternating monthly cycles of tamoxifen (TAM) and estradiol during which sequential (24-hr) methotrexate, 5-fluorouracil, and leucovorin are administered at 2-week intervals. Twenty-five patients with metastatic breast cancer received greater than 80 endocrine cycles and greater than 300 courses of chemotherapy by this protocol; two-thirds of these patients had previously failed other endocrine or chemotherapy regimens. Most patients experienced grade 1 or 2 myelosuppression or gastrointestinal symptoms during at least one treatment cycle; however, overall toxicity was considered to be mild and therapy was very well tolerated. Serum levels of estradiol, estrone, TAM, and TAM metabolites were measured during all phases of the endocrine cycle in five patients on chronic therapy. Concentrations of TAM and its more abundant metabolite, N-desmethyltamoxifen (N-desTAM), rose twofold during antiestrogen therapy and fell during estrogen priming, with mean levels persisting greater than 100 ng/ml throughout the priming interval. Mean estradiol (E2) and estrone (E1) levels rose during priming and were sustained fivefold and tenfold above the basal postmenopausal levels measured during antiestrogen treatment. Calculating the serum molar ratios of [TAM + N-desTAM]/[E2 + E1] during each phase of the treatment cycle confirmed that estrogen priming was achieved pharmacologically by this endocrine schedule. Clinical remissions were observed in this small patient sample with seven of 18 patients achieving either complete (28%) or partial (11%) responses, and an additional 39% obtaining disease stabilization. Further clinical study is necessary to evaluate the optimal response rate of this regimen and to determine whether cyclic estrogen priming by this schedule results in enhanced tumor cell proliferation in vivo.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Estradiol; Estrone; Female; Fluorouracil; Humans; Kinetics; Leucovorin; Methotrexate; Middle Aged; Tamoxifen

A method for the analysis of tamoxifen and its metabolites in plasma from tamoxifen treated breast cancer patients, by capillary GC-MS using selected ion monitoring has been developed. Metabolite extraction was carried out on a Sep-pak C18 cartridge and metabolite purification by selective ion exchange chromatographic steps. Satisfactory recovery of radioactive standards through the extraction and purification steps was obtained. The method was shown to be accurate and precise with precision coefficient of variation values ranging from 4.3-11% for tamoxifen and its metabolites. Tamoxifen, 4-hydroxytamoxifen, metabolite Y and N-desmethyltamoxifen were identified with certainty in patient plasma on the basis of GC relative retention times and mass spectral comparison with authentic standards; because of their low abundance in plasma cis-metabolite E and 3,4-dihydroxytamoxifen could only be tentatively identified but identical GC behaviour and a satisfactory comparison of the abundance of key fragment ions was achieved. The tamoxifen and metabolite concentration ranges (ng X ml-1) in the group of patients who received 40 or 80 ng tamoxifen for 14 days were tamoxifen, 307-745; N-desmethyltamoxifen, 185-491; 4-hydroxytamoxifen, 1.4-2.5; 3,4-dihydroxytamoxifen, 0.7-2.0; metabolite Y, 19.0-112; and metabolite E1, 0.9-2.0.

Topics: Aged; Aged, 80 and over; Breast Neoplasms; Female; Gas Chromatography-Mass Spectrometry; Humans; Middle Aged; Quality Control; Tamoxifen

Recent biochemical and pharmacological findings concerning tamoxifen (TMX) have proven that both the unchanged drug and the main metabolites, N-desmethyltamoxifen (NDT) and 4-hydroxytamoxifen (4OHT) are biologically active. An HPLC method based on on-line post-column UV irradiation with fluorescence detection is described. Optimized conditions allowed complete and rapid separation of TMX 4OHT, NDT and two other recently reported metabolites, Y and Z. This method was applied to plasma and cytosol drug and metabolite analyses. In plasma, from the moment of initial drug administration until the steady state (after 1 month or more of continuous oral TMX treatment), the values of NDT to TMX ratios were completely reversed: 22 to 215 in mean %, P less than 0.01. The presence of metabolites Y and Z is significant. 4OHT, hardly detectable at the first dose, was measured at the steady state with high interpatient variability. It is hypothesized that metabolite evolution with time may be due to auto-induction of drug metabolism. In cytosols, which were all obtained during continuous TMX treatment, the ratios between TMX and metabolites were comparable to those observed in plasma, but with greater interpatient variability. Metabolite Y was not detectable in cytosols. This variability was not linked to the levels of cytosolic oestradiol receptors before initiation of treatment.

Topics: Breast Neoplasms; Chromatography, High Pressure Liquid; Cytosol; Female; Humans; Methods; Tamoxifen

Tamoxifen, 4-hydroxytamoxifen and desmethyltamoxifen levels were measured in cytosolic and 0.5 M KCl extracted nuclear fractions from a small series of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM). Tamoxifen and desmethyltamoxifen were the most abundant metabolites. There was a small increment in the relative abundance of 4-hydroxytamoxifen in the nuclear extract over cytosol relative to both tamoxifen and desmethyltamoxifen. Further, there was a selective retention of tamoxifen relative to desmethyltamoxifen in the nuclear extract relative to the cytosol. It is concluded that all three compounds could potentially contribute to estrogen receptor mediated antiestrogenic effects in this target tissue.

Topics: Breast Neoplasms; Cell Nucleus; Cytosol; Female; Gas Chromatography-Mass Spectrometry; Humans; Potassium Chloride; Tamoxifen

The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor. Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth. In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor. In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected.

Topics: Breast Neoplasms; Cell Division; Cells, Cultured; Clomiphene; Enclomiphene; Estrogen Antagonists; Female; Humans; Receptors, Estrogen; Tamoxifen

Topics: Breast Neoplasms; Cell Cycle; Cell Line; Depression, Chemical; Humans; Tamoxifen

A new hydroxylated metabolite of tamoxifen, Metabolite Y [trans-1-(p-beta-hydroxyethoxyphenyl)-1,2-diphenylbut-1-ene] was characterized and subsequently measured by high-performance liquid chromatography in serum from patients receiving normal (10 mg twice daily) and high dose (greater than or equal to 150 mg twice daily) tamoxifen therapy for treatment of advanced breast cancer. In normal-dose patients, the serum level of Metabolite Y ranged between 6 and 60 ng/ml. This contrasted with serum levels of 80 to 180 ng/ml for tamoxifen and 200 to 300 ng/ml for N-desmethyltamoxifen, the major metabolite of tamoxifen. Serum levels of all three components were unchanged in one patient during the 24 hr after the cessation of tamoxifen therapy. Maximum serum levels of Metabolite Y were 800 ng/ml with concentrations of 1 micrograms/ml for tamoxifen and 2 micrograms/ml for N-desmethyltamoxifen in a patient on a 2-year course of high-dose therapy. Metabolite Y inhibited the binding of 17 beta-[3H]-estradiol to rat uterine and human breast carcinoma estrogen receptor. However, this metabolite was only weakly active: monohydroxytamoxifen [relative binding affinity (RBA) = 280]; tamoxifen (RBA = 6); Metabolite E (RBA = 3); N-desmethyltamoxifen (RBA = 4); Metabolite Y (RBA = 0.5). In 3-day immature rat uterine weight tests, Metabolite Y was a partial agonist with weak antiestrogenic activity. Although Metabolite Y has only weak activity, this compound would be expected to contribute to the overall antiestrogenic and antitumor properties of tamoxifen during therapy.

Topics: Animals; Biological Assay; Breast Neoplasms; Chromatography, High Pressure Liquid; Estradiol; Female; Humans; Rats; Tamoxifen; Uterus

Topics: Breast Neoplasms; Estradiol; Female; Gas Chromatography-Mass Spectrometry; Humans; Receptors, Estrogen; Tamoxifen

Topics: Breast Neoplasms; Bromocriptine; Drug Therapy, Combination; Estradiol; Female; Half-Life; Humans; Hydrocortisone; Middle Aged; Tamoxifen

Serum concentrations of tamoxifen and its metabolite, N-desmethyltamoxifen (DMT) were determined in six post-menopausal patients with advanced breast cancer. Following a single 10-mg dose PO parent drug was detected in the serum, with a peak concentration of 17.5 ng/ml. Concentrations of the N-desmethyl metabolite were below the limit of detection (less than 2.5 ng/ml). After 21 days' oral therapy with 10 mg b.i.d. the serum concentration of tamoxifen had increased ten fold, while DMT was now present in comparable amounts. Two patients were further studied for a longer time period. There was little change in the serum concentration of tamoxifen, while the DMT increased two fold above its value at 21 days.

Topics: Breast Neoplasms; Female; Humans; Middle Aged; Tamoxifen