n-desmethyltamoxifen and afimoxifene

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

Potential drugdrug interactions are a concern for patients taking tamoxifen.. This study was designed to determine the effect of coadministering desvenlafaxine on tamoxifen pharmacokinetics.. This open-label, 2-period inpatient and outpatient study enrolled healthy, postmenopausal women. Period 1, day 1, subjects were administered tamoxifen 40 mg followed by 23 days of blood sampling for pharmacokinetic analyses. During period 2, subjects received desvenlafaxine 100 mg/d for 28 days; a single dose of tamoxifen 40 mg was administered with desvenlafaxine 100 mg on day 7, followed by 23 days of blood sampling. Pharmacokinetics of tamoxifen and its metabolites (AUC over infinite time (AUC(inf)), AUC to the last measurable concentration (AUC(last)), peak plasma concentration (C(max)) were compared for monotherapy vs. combination therapy using the ratio of adjusted mean differences. A superposition method was used in the statistical analysis of N-desmethyl-tamoxifen and endoxifen to address the carry-over observed for those metabolites. The test for interaction was considered negative if the 90% confidence intervals (CIs) for the ratios were within 80 - 125%.. Coadministration of tamoxifen with steady-state desvenlafaxine did not alter tamoxifen AUC(inf), AUC(last), and C(max), as reflected by the ratio of adjusted geometric means (90% CIs) of 100.7% (96.7%, 104.9%), 103.5% (100.2%, 106.9%), and 99.4% (94.0%, 105.2%), respectively. Similarly, coadministration did not alter 4-hydroxy- tamoxifen and N-desmethyl-amoxifen pharmacokinetics. The 11.8% (88.2% (82.6%, 94.2%)) and 8.0% (92.0% (84.7%, 100.0%)) decreases in endoxifen AUC(last) and C(max), respectively, were not significant (90% CIs fell wholly within the prespecified acceptance range).. Steady-state desvenlafaxine 100 mg did not affect tamoxifen pharmacokinetics. For women treated with tamoxifen, desvenlafaxine may represent a safe and effective treatment unlikely to alter tamoxifen efficacy.

Topics: Area Under Curve; Desvenlafaxine Succinate; Drug Interactions; Female; Humans; Middle Aged; Postmenopause; 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

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

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, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug-drug-interactions.

Topics: Alkenes; Cell Line; Cytochrome P-450 Enzyme System; Estrogens; Gene Expression Regulation, Enzymologic; Glucuronides; Glucuronosyltransferase; Hepatocytes; Humans; Microsomes, Liver; Phenols; Tamoxifen

Tamoxifen is an endocrine-active pharmaceutical (EAP) that is used world-wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically-active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen-treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish (Tautogolabrus adspersus). In plasma from tamoxifen-exposed cunner, the predominant metabolite was 4-hydroxytamoxifen, while in rats it was N-desmethyltamoxifen. Because 4-hydroxytamoxifen is a more biologically active metabolite than N-desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts demonstrates that tamoxifen alters the biochemical profile in this nontarget species. Understanding the consequence of tamoxifen exposure in nontarget species, and assessing the discrepancies between sex- and species-mediated endpoints, is a step toward understanding how to accurately assess the risks posed by EAPs, such as tamoxifen, in the aquatic environment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1834-1850, 2016.

Topics: Animals; Biological Assay; Eggs; Environmental Exposure; Female; Microsomes, Liver; Perciformes; Reproduction; Selective Estrogen Receptor Modulators; Tamoxifen; Water Pollutants, Chemical

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

Tamoxifen is successfully used for both treatment and prevention of estrogen-dependent breast cancer, yet side effects and development of resistance remain problematic. Endoxifen is a major active metabolite of tamoxifen that is being investigated for clinical use. We hypothesized that endoxifen and perhaps other major metabolites of tamoxifen may affect the ability of human estrogen sulfotransferase 1E1 (hSULT1E1) and human phenol sulfotransferase 1A1 isoform 1 (hSULT1A1*1) to catalyze the sulfation of estradiol, an important mechanism in termination of estrogen signaling through loss of activity at estrogen receptors. Our results indicated that endoxifen, N-desmethyltamoxifen (N-desTAM), 4-hydroxytamoxifen (4-OHTAM), and tamoxifen-N-oxide were weak inhibitors of hSULT1E1 with Ki values ranging from 10 μM to 38 μM (i.e., over 1000 times higher than the 8.1 nM Km value for estradiol as substrate for the enzyme). In contrast to the results with hSULT1E1, endoxifen and 4-OHTAM were significant inhibitors of the sulfation of 2.0 µM estradiol catalyzed by hSULT1A1*1, with IC50 values (9.9 μM and 1.6 μM, respectively) that were similar to the Km value (1.5 μM) for estradiol as substrate for this enzyme. Additional investigation of the interaction of these metabolites with the two sulfotransferases revealed that endoxifen, 4-OHTAM, and N-desTAM were substrates for hSULT1E1 and hSULT1A1*1, although the relative catalytic efficiencies varied with both the substrate and the enzyme. These results may assist in future elucidation of cell- and tissue-specific effects of tamoxifen and its metabolites.

Topics: Antineoplastic Agents, Hormonal; Arylsulfotransferase; Drugs, Investigational; Enzyme Inhibitors; Estradiol; Humans; Kinetics; Recombinant Proteins; Substrate Specificity; Sulfotransferases; Tamoxifen

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

The aim of this study was to validate an earlier developed high-performance highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for quantification of tamoxifen and its three main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen) in scalp hair. This non-invasive method might, by segmental analysis of hair, be useful in the determination of the concentration of drugs and its metabolites over time, which can be used to study a wide variety of clinical relevant questions. Hair samples (150-300 hair strands, cut as close to the scalp as possible from the posterior vertex region of the head) were collected from female patients taking tamoxifen 20mg daily (n=19). The analytes were extracted using a liquid-liquid extraction procedure with carbonate buffer at pH 8.8 and a mixture of n-hexane/isopropranol method, followed by UPLC-MS/MS chromatography, based on an earlier validated method. The calibration curves were linear in the range of 1.00-200 pmol for tamoxifen and N-desmethyl-tamoxifen, with lower limit of quantitation of 1.00 pmol and 0.100-20.0 pmol with lower limit of quantitation of 0.100 pmol for endoxifen and 4-hydroxy-tamoxifen. Assay performance was fair with a within-run and between-run variability less than 9.24 at the three quality control samples and less than 15.7 for the lower limit of quantitation. Importantly, a steep linear decline was observed from distal to proximal hair segments. Probably, this is due to UV exposure as we showed degradation of tamoxifen and its metabolites after exposure to UV-light. Furthermore, higher concentrations of tamoxifen were found in black hair samples compared to blond and brown hair samples. We conclude that measurement of the concentration of tamoxifen and its main metabolites in hair is possible, with the selective, sensitive, accurate and precise UPLC-MS/MS method. However, for tamoxifen, it seems not possible to determine exposure over time with segmental analysis of hair, probably largely due to the effect of UV irradiation. Further research should therefore focus on quantification of other anticancer drugs, in segmented scalp hair, that are less sensitive to UV irradiation.

Topics: Adult; Aged; Calibration; Chromatography, High Pressure Liquid; Female; Hair; Humans; Hydrogen-Ion Concentration; Light; Limit of Detection; Middle Aged; Quality Control; Reproducibility of Results; Scalp; Tamoxifen; Tandem Mass Spectrometry; Ultraviolet Rays

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

A highly sensitive HPLC-UV method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation on a Hypersil Gold(®) C18 column (150mm×4.6mm, 5μm) with isocratic elution. Mobile phase was a mixture of triethylammonium phosphate buffer 5mM pH 3.3 and acetonitrile (57:43, v/v). Total analytical run time was 16min. Precision assays showed CV % lower than 10.53% and accuracy in the range of 93.0-104.2%. The lower limits of quantification (0.75-8.5ngml(-1)) are adequate to measure clinically relevant concentrations in plasma samples. The method was successfully applied to 110 clinical plasma samples. Median plasma levels and interquartile range were: tamoxifen 55.77ngml(-1) (38.42-83.69ngml(-1)), N-desmethyltamoxifen 124.83ngml(-1) (86.81-204.80ngml(-1)), 4-hydroxytamoxifen 1.09ngml(-1) (0.76-1.53ngml(-1)) and endoxifen 6.18ngml(-1) (4.17-8.22ngml(-1)). The procedure has adequate analytical performance and can be employed in therapeutic drug monitoring of tamoxifen or pharmacokinetics studies.

Topics: Antineoplastic Agents, Hormonal; Chromatography, High Pressure Liquid; Drug Monitoring; Female; Humans; Limit of Detection; Sensitivity and Specificity; Tamoxifen

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Topics: Animals; Antineoplastic Agents, Hormonal; Aorta, Thoracic; Estradiol; Fulvestrant; Indomethacin; Male; NG-Nitroarginine Methyl Ester; Prostaglandins; Rats; Rats, Wistar; Tamoxifen; Vasodilation

Tamoxifen has been suggested to produce beneficial cardiovascular effects, although the mechanisms for these effects are not fully known. Moreover, although tamoxifen metabolites may exhibit 30-100 times higher potency than the parent drug, no previous study has compared the effects produced by tamoxifen and its metabolites on vascular function. Here, we assessed the vascular responses to acetylcholine and sodium nitroprusside on perfused hindquarter vascular bed of rats treated with tamoxifen or its main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen, and endoxifen) for 2 weeks. Plasma and whole-blood thiobarbituric acid reactive substances (TBARS) concentrations were determined using a fluorometric method. Plasma nitrite and NOx (nitrite + nitrate) concentrations were determined using an ozone-based chemiluminescence assay and Griess reaction, respectively. Treatment with tamoxifen reduced the responses to acetylcholine (pD(2) = 2.2 +/- 0.06 and 1.9 +/- 0.05 after vehicle and tamoxifen, respectively; P < 0.05), while its metabolites improved these responses (pD(2) = 2.5 +/- 0.04 after N-desmethyl-tamoxifen, 2.5 +/- 0.03 after 4-hydroxy-tamoxifen, and 2.6 +/- 0.08 after endoxifen; P < 0.01). Tamoxifen and its metabolites showed no effect on endothelial-independent responses to sodium nitroprusside (P > 0.05). While tamoxifen treatment resulted in significantly higher plasma and whole blood lipid peroxide levels (37% and 62%, respectively; both P < 0.05), its metabolites significantly decreased lipid peroxide levels (by approximately 50%; P < 0.05). While treatment with tamoxifen decreased the concentrations of markers of nitric oxide formation by approximately 50% (P < 0.05), tamoxifen metabolites had no effect on these parameters (P > 0.05). These results suggest that while tamoxifen produces detrimental effects, its metabolites produce counteracting beneficial effects on the vascular system and on nitric oxide/reactive oxygen species formation.

Topics: Animals; Blood Pressure; Endothelium, Vascular; Heart Rate; Male; Nitric Oxide; Oxidative Stress; Perfusion; Rats; Rats, Wistar; Tamoxifen; Thiobarbituric Acid Reactive Substances; Vascular Resistance

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

To characterize the interindividual variability and the individual CYP involved in the formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen from tamoxifen.. Microsomes from 50 human livers were used to characterize the interindividual variability in the alpha-hydroxylation, N-desmethylation and N-didesmethylation of tamoxifen. Selective inhibitors and recombinant enzymes were used to identify the forms of CYP catalysing these reactions.. The rates of formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen were highly variable, and correlated with each other (P < 0.0001). The respective ranges were 0.7-11.4, 25.7-411, and below the limit of quantification--4.4 pmol mg(-1) protein min(-1). Formation of all metabolites was observed with expressed recombinant CYP3A4, inhibited by troleandomycin (65, 77 and 35%, respectively, P < 0.05) and associated with CYP3A4 expression (rs = 0.612, rs = 0.585 and rs = 0.430, P < 0.01, respectively).. Formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen in vitro is highly variable and mediated predominantly by CYP3A4.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Female; Genetic Variation; Humans; Male; Microsomes, Liver; Middle Aged; 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

Topics: Antidepressive Agents, Second-Generation; Antineoplastic Agents, Hormonal; Estrogen Receptor Modulators; Female; Humans; Paroxetine; Selective Serotonin Reuptake Inhibitors; Tamoxifen

The effects of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen on transport attributable to P-glycoprotein were studied using Caco-2 cell monolayers in a transwell system, with rhodamine-123 as an index substrate for inhibition studies. The three compounds did not demonstrate differential flux between basal-apical and apical-basal directions in Caco-2 monolayers. The mean IC50 values for inhibition of rhodamine-123 transport were: 29 microM for tamoxifen; 26 microM for N-desmethyltamoxifen; and 7.4 microM for 4-hydroxytamoxifen. The three compounds were also evaluated as potential inhibitors of human CYP3A based on an in vitro model using triazolam hydroxylation by human liver microsomes as an index reaction. Mean (+/-SE) IC50 values versus formation of alpha-hydroxy-triazolam and 4-hydroxy-triazolam in human liver microsomes were, respectively: 23.5 (+/-3.9) and 18.4 (+/-5.3) microM for tamoxifen; 10.2 (+/-1.7) and 9.2 (+/-1.5) microM for N-desmethyltamoxifen; and 2.6 (+/-0.5) and 2.7 (+/-0.3) microM for 4-hydroxytamoxifen. Thus, tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, do not appear to be substrates for transport by P-glycoprotein. However, tamoxifen has the potential to inhibit transport mediated by P-glycoprotein as well as CYP3A-mediated metabolism. Inhibitory effects of the principal metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen, may exceed those of the parent drug. Tamoxifen, and possibly its metabolites, may have the potential to cause drug interactions by inhibiting both drug transport and metabolism. This possibility requires further evaluation in clinical studies.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Caco-2 Cells; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Humans; Tamoxifen

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.

Topics: Biotransformation; Culture Techniques; Cytochrome P-450 Enzyme System; DNA Adducts; Endometrium; Female; Humans; Immunohistochemistry; Recombinant Proteins; Tamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Topics: Antineoplastic Agents, Hormonal; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Humans; Hydroxylation; In Vitro Techniques; Isoenzymes; Microsomes, Liver; Stereoisomerism; Tamoxifen

This study investigates which CYP forms are responsible for the conversion of tamoxifen to its putative active metabolite alpha-hydroxytamoxifen and irreversible binding to DNA. We have used eight different baculovirus expressed recombinant human CYP forms and liquid chromatography-mass spectrometry to show that only CYP3A4 is responsible for the NADPH-dependent alpha-hydroxylation of tamoxifen. Surprisingly, this CYP did not catalyse the formation of 4-hydroxytamoxifen. We demonstrate for the first time, by means of accelerator mass spectrometry, that CYP3A4 also catalysed the activation of [(14)C]tamoxifen to intermediates that irreversibly bind to exogenous DNA. Incubation of [(14)C]tamoxifen (20.6 kBq, 100 micro M) with CYP3A4, in the presence of NADPH for 60 min led to levels of DNA binding of 39.0+/-9.0 adducts/10(8) nucleotides (mean +/- SE, n = 6). While CYP3A4 converted tamoxifen to N-desmethyltamoxifen (38.3 +/- 7.20 pmol/20 min/pmol CYP, n = 4), the polymorphic CYP2D6 showed the highest activity for producing this metabolite (48.6+/-1.52pmol/20 min/pmol CYP). CYP2D6 was also the most active in catalysing 4-hydroxylation of tamoxifen, although an order of magnitude lower level was also detected with CYP2C19. With tamoxifen as substrate, no 3,4-dihydroxytamoxifen could be detected with any CYP form. CYP2B6 did not catalyse the metabolism or the binding of tamoxifen to DNA. It is concluded that CYP3A4 is the only P450 of those tested that converts tamoxifen to alpha-hydroxytamoxifen and the only one that results in appreciable levels of irreversible binding of tamoxifen to DNA.

Topics: Biotransformation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DNA; Humans; Hydroxylation; Isoenzymes; NADP; Prodrugs; Tamoxifen

Tamoxifen (TAM), an antiestrogen, has been approved for use by women at risk for developing hormone-dependent breast cancer. Administration of TAM to pregnant CD-1 mice apparently results in reproductive tract toxicity in female offspring. However, there is little or no data describing potential TAM-induced fetal toxicity to women who may become pregnant while receiving prophylactic TAM treatment. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, the present work describes a capillary electrophoresis (CE)-based analytical technique used for detection of TAM and two major metabolites, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in CD-1 mouse fetal tissue. TAM-derived material was extracted from CD-1 mouse fetuses 2-12 h following TAM administration (100 mg/kg) to dams on gestation day 16. The presence of TAM, DMT, and 4-HT was confirmed in the solvent extracts by nonaqueous CE. The limit of detection of TAM by UV absorption was approximately 675 amol at a signal-to-noise ratio of 2:1. This work demonstrates both transplacental transport of TAM in CD-1 mice and a sensitive analytical technique for detecting low concentrations of TAM and similar compounds in biological tissues.

Topics: Animals; Electrophoresis, Capillary; Female; Fetus; Mice; Pregnancy; Tamoxifen

The effects of chronic oral administration of tamoxifen citrate, at a dose of 0.4 mg/kg/day, were compared to those of subcutaneous (s.c) administration of tamoxifen citrate, 4-hydroxy tamoxifen, N-desmethyl tamoxifen and intermittent oral tamoxifen administration on the fertility of the male rat and its post reversal progeny. The fertility parameters of 120 day-treated male rat sires from all groups and post reversal male F1 progeny of tamoxifen-treated sires were assessed. Chronic tamoxifen treatment via oral or s.c. routes reduced the fertility of the male rat, weights of accessory sex glands, serum luteinizing hormone, and testosterone levels without altering potency or sperm counts. However, antifertility effects of s.c. treatment were comparatively more consistent than those of oral treatment. 4-hydroxy and N-desmethyl tamoxifen failed to produce significant antifertility effects in the male rat. The antifertility effects of intermittent oral treatment were more sustained than those of chronic oral tamoxifen treatment. It is inferred that hepatic metabolism of tamoxifen interferes with its antifertility effects via oral route and that the parameters affected by chronic oral exposure in the male sires are completely reversed in progeny ensuing after an adequate period of drug withdrawal.

Topics: Administration, Oral; Animals; Estradiol; Female; Fertility; Follicle Stimulating Hormone; Genitalia, Male; Infertility, Male; Luteinizing Hormone; Male; Organ Size; Rats; Tamoxifen; Testosterone

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

The metabolism of tamoxifen was studied in female Sprague-Dawley rat and mouse liver slices and homogenates, and the three principal tamoxifen metabolites, 4-hydroxytamoxifen, N-desmethyl-tamoxifen and tamoxifen N-oxide, were identified by HPLC using authentic standards. It was not possible to identify any of the minor metabolites such as the epoxides using this technique. The N-oxide metabolite only appeared when NADPH was added to the system; this is because the production of tamoxifen N-oxide is primarily mediated by microsomal flavin monooxygenase (FMO) which is NADPH dependent. However, this metabolite did appear in incubations with mouse liver slices only, because they are rich in flavin monooxygenases (FMOs). It did not appear in female rat or mouse liver homogenates, because the NADPH present is destroyed during homogenisation, therefore it was necessary to add NADPH to the system to produce the N-oxide metabolite. The purpose of this study was to investigate the effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates. Female rat liver slices and homogenates were incubated with the following inhibitors (1 mM): cimetidine, ascorbate, sodium azide and reduced glutathione. Cimetidine, a general P-450 inhibitor, inhibited the production of the N-desmethyl metabolite by about 80%; this is in agreement with the action of the other inhibitors. Reduced glutathione, ascorbate and sodium azide are mainly peroxidase inhibitors, so therefore from these novel and interesting results it was possible to suggest that peroxidases play a role in the metabolism of tamoxifen. This observation was also strengthened when the production of the N-desmethyl metabolite increased when horseradish peroxidase was added to the incubate. The production of 4-hydroxytamoxifen was reduced and the N-oxide metabolite was completely inhibited in the presence of peroxidase inhibitors. When rat liver homogenates was incubated with superoxide dismutase (SOD) and catalase, it was observed that the N-desmethyl metabolite disappeared completely at 60 min and the N-oxide and 4-hydroxy metabolites were completely inhibited. However, this phenomenon was only observed when SOD and catalase were preincubated for 30 min with the rat liver homogenate at 37 degrees C; without preincubation the production of these metabolites was unaffected. Finally, the effect of long incubation periods (300 min) on the production of metabolites was examined.

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Cimetidine; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Female; Glutathione; In Vitro Techniques; Liver; Mice; NADP; Rats; Sodium Azide; Tamoxifen; Time Factors

The literature concerning cytoskeletal changes and metastatic progression is unresolved, some studies suggesting a positive association between the ability of cells to organize their cytoskeleton and others suggesting an inverse correlation. In an attempt to learn more about cytoskeletal changes and the ability of melanoma cells to interact with extracellular matrix proteins we examined the effects of pharmacological manipulation of cell attachment and cell invasion through fibronectin on levels of F-actin and vimentin in a highly metastatic cutaneous melanoma cell line (A375-SM cells). Additionally, we examined whether any correlation existed between the levels of the cytoskeletal proteins and subpopulations of the cell line of varying invasive ability. We report that agents which reduced cell attachment to plastic and invasion through fibronectin in vitro (tamoxifen, N-desmethyltamoxifen and 17beta-oestradiol) caused increases in levels of F-actin and vimentin, whereas agents which did not affect attachment or invasion (4-hydroxytamoxifen and dihydrotestosterone) had little or no effect on the cytoskeletal proteins. In contrast, however, those cells which were most effective at invading through fibronectin were significantly better at acutely increasing their levels of F-actin and vimentin than less invasive cells. We speculate that the ability to rapidly and possibly reversibly alter the cytoskeleton might be associated with metastatically successful cells in vivo.

Topics: Actins; Antineoplastic Agents, Hormonal; Cell Adhesion; Cytoskeletal Proteins; Cytoskeleton; Dihydrotestosterone; Embryonal Carcinoma Stem Cells; Estradiol; Fibronectins; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neoplastic Stem Cells; Skin Neoplasms; Tamoxifen; Tumor Cells, Cultured; Vimentin

Tamoxifen (TAM), an antiestrogen, is widely used to treat hormone-dependent breast cancer in post-menopausal women. TAM may be used as a chemopreventive agent in women of child-bearing age; however, few data exist describing potential TAM-induced fetal toxicity. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, this work describes an analytical technique utilizing capillary electrophoresis (CE) for the detection of circulating levels of TAM, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in maternal rodent serum. Greater than 90% of 3H-labeled TAM was extractable from serum using 98:2 hexane-isoamyl alcohol. Optimum separation of TAM, DMT, and 4-HT was obtained on a 57 cmx50 microm capillary using a nonaqueous buffer system of 1:1 methanol-acetonitrile containing 50 mM ammonium acetate and 1% acetic acid. 4-Dimethylaminopyridine was used as internal standard. Temperature and voltage were optimized at 40 degrees C and 15 kV, respectively. The limit of detection of TAM by UV detection at 214 nm was approximately 800 amol. TAM and DMT were confirmed in serum of female rats 4 h following a single oral dose of 120 mg/kg. Transplacental exposure of TAM to fetal tissue will be evaluated using this technique.

Topics: Animals; Anticarcinogenic Agents; Buffers; Electrophoresis, Capillary; Female; Rats; Rats, Inbred F344; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Tamoxifen

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, an antiestrogen used in the treatment of breast cancer, was assessed for carcinogenic potential in the two-stage model of experimental hepatocarcinogenesis. Groups of female Fisher F344 rats were initiated with a non-necrogenic, subcarcinogenic dose of diethylnitrosamine (DEN; 10 mg/kg, po) and fed tamoxifen at a concentration of 250 mg per kg of AIN-76A diet for 6 or 15 months. The livers of these animals exhibited an increase in size and number of altered hepatic foci compared with those animals which were initiated with DEN but not exposed to tamoxifen. This finding indicates that tamoxifen may have a carcinogenic potential in the rat liver. After 6 months of treatment, neoplastic nodules were observed in 3/8 rats in the DEN-initiated, tamoxifen-treated group. In the initiated group provided with tamoxifen for 15 months, neoplastic nodules were observed in 7/8 rats and hepatocellular carcinomas in 3/8 rats. The serum level of tamoxifen in these rats was 200-300 ng/ml. The ratio of tamoxifen, 4-hydroxy tamoxifen, and N-desmethyl tamoxifen was 1:0.1:0.5-1 in the serum. When adjusted for age-related weight increases, the serum and liver levels of tamoxifen and its N-desmethyl metabolite did not change over the 15 months. In the rat liver, the level of tamoxifen and its N-desmethyl metabolite was 10-29 micrograms/g liver after 6 or 15 months of chronic dietary administration. The ratio of tamoxifen:4-hydroxy tamoxifen:N-desmethyl tamoxifen was 1:0.1.3-3.3 in the liver. Therefore, the liver had 20- to 30-fold more tamoxifen and 4-hydroxy tamoxifen and at least 100-fold more N-desmethyl tamoxifen than the serum (assuming 1 gram of tissue is equivalent to 1 ml of serum). These results indicate that tamoxifen is a promoting agent for the rat liver at serum levels found in patients given the usual therapeutic course of tamoxifen. The high concentrations of tamoxifen attained in the rat liver indicate that actions other than its known estrogenicity for liver could contribute to its promoting action. In addition, these results indicate that the pharmacodynamic differences in tamoxifen metabolism in rats and humans and at low versus high doses should be determined. Thus, the therapeutic indications for tamoxifen should be balanced by the potential risk it may present as a promoting agent in mammalian liver.

Topics: Animals; Carcinogens; Diet; Diethylnitrosamine; Female; Liver; Liver Neoplasms, Experimental; Rats; Rats, Inbred F344; Tamoxifen; Time Factors

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

A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 x 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 x 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.

Topics: Adolescent; Adult; Chromatography, High Pressure Liquid; Humans; Sensitivity and Specificity; Tamoxifen

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

Screening studies of tamoxifen (TAM) have shown that only one culture, Streptomyces rimosus ATCC 2234 was able to metabolize tamoxifen to 4-hydroxy tamoxifen (OHT) which was obtained in a 10% yield and chemically identified by 2 D nmr spectroscopy. A number of microorganisms were capable of biotransforming TAM to tamoxifen-N-oxide (TNO), and desmethyltamoxifen (DMT). The chemical identity of the isolated metabolites was achieved using gas chromatography/mass spectrometry and different nmr spectroscopic techniques.

Topics: Bacteria; Biotransformation; Fungi; Tamoxifen

Presence of a collagenolytic activity has been demonstrated in the human leukemic cell line K562. Among various effectors studied, tamoxifen, a well-known antiestrogenic compound, exhibited a strong inhibitory effect. After 3 days of culture in the presence of 10(-7) M of tamoxifen, 75% of the collagenolytic activity was inhibited. Hydroxytamoxifen and N-desmethyltamoxifen were equally potent inhibitors though devoid of the direct cytotoxic effect. Cis-tamoxifen was less efficient. K562 cells have no binding sites for estrogens but they possess high affinity binding sites for 3H-tamoxifen (295 fmol/mg of proteins, KD = 0.25 x 10(-9) M). Tamoxifen had no effect on cellular differentiation or enzyme secretion. Anticollagenolytic activity of tamoxifen (10(-7)-10(-6) M) could be related to its inhibitory action on plasmin and plasminogen activator.

Topics: Binding Sites; Collagen; Estrogen Antagonists; Fibrinolysin; Humans; Leukemia; Plasminogen Inactivators; Tamoxifen; 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

A new and simplified high-performance liquid chromatographic (HPLC) method for the determination of the antiestrogenic drug tamoxifen (TAM) and its desmethylated and hydroxylated metabolites in human plasma is described. Specific and sensitive fluorescence detection is achieved by "on-line" photochemical conversion of the TAM structure to a highly fluorescent phenanthrene product using a newly developed post-column photoreactor included in the HPLC system. A highly selective chromatographic separation was established by using unmodified silica with aqueous mobile phase for separation, with the sample preparation step on a small CN-propyl pre-column, included in the HPLC system. Due to the high specificity of the separation and detection system, even small volumes of deproteinized plasma sample can be injected directly without prior sample extraction. The described method permits a very fast and reproducible determination of TAM and its two major metabolites in plasma on a routine basis, down to 100 pg ml-1 concentration.

Topics: Chromatography, High Pressure Liquid; Humans; Indicators and Reagents; Photochemistry; Spectrometry, Fluorescence; Tamoxifen

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 biological and morphological effects of cis-tamoxifen, N-desmethyltamoxifen and 4-hydroxy-tamoxifen, administered sc alone (100 micrograms/animal) or combined with estradiol (20 micrograms/animal) were studied in the uterus and vagina of the guinea pig. After 2 days treatment the values of the uterine wet weights (mg +/- SD of 6-10 animals in each study) were as follows: non-treated animals (control): 142 +/- 15; animals treated with cis-tamoxifen: 119 +/- 4; N-desmethyltamoxifen: 280 +/- 20; 4-hydroxytamoxifen: 268 +/- 25. The values after long treatment were: 177 +/- 30; 490 +/- 65; 394 +/- 36 and 581 +/- 60, respectively. After short treatment the weights of the vaginas were: control: 99 +/- 20; cis-Tamoxifen: 67 +/- 2; N-desmethyltamoxifen: 153 +/- 25; 4-hydroxytamoxifen: 166 +/- 7; and after the long treatment: 155 +/- 40; 660 +/- 41; 467 +/- 38 and 502 +/- 61, respectively. N-desmethyltamoxifen and 4-hydroxytamoxifen increased the progesterone receptors in the uterus after short treatment (P less than 0.01) but not after 12 days treatment. On the other hand, there was no effect on progesterone receptor in the vagina after the short treatment but a very stimulatory effect after the long treatment. The morphological alterations after 12-days treatment indicate that the three tamoxifen derivatives in the two tissues studied provoke intense alterations in different organelles. In conclusion, it is suggested that the tamoxifen derivatives can act as real agonists in the uterus and vagina of the newborn guinea pig, and they do not block the effect provoked by estradiol.

Topics: Animals; DNA; Estradiol; Female; Guinea Pigs; Receptors, Progesterone; Tamoxifen; Time Factors; Uterus; Vagina

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

As previously reported, ovarian epithelial carcinomas may respond to endocrine therapy. We examined the direct effect of progesterone, medroxyprogesteroneacetate, gestoneron, 17-beta-estradiol, tamoxifen, 4-OH-tamoxifen, or N-desmethyltamoxifen on the proliferative capacity of ovarian carcinoma cells by means of the colony assay described by Hamburger and Salmon. The growth rate of 25 tested tumors (ascitic fluid, primary tumor, metastases) was 68%. The plating efficiency was 0.078%. Beside the drug testing estrogen and progesterone receptor levels were determined. The inhibition of colony survival was slightest with 17-beta-estradiol, more pronounced with medroxyprogesteroneacetate, gestoneron, N-desmethyltamoxifen, and progesterone, and greatest with 4-OH-tamoxifen and tamoxifen. Significant and dose-dependent inhibition of greater than 70% was observed with tamoxifen and 4-OH-tamoxifen in 80% of the tested tumors. There was no significant correlation between the in vitro responsiveness and the level of hormonal act not only via an estrogen receptor but also via an antiestrogen-binding site.

Topics: Adenocarcinoma; Aged; Cell Division; Cells, Cultured; Estradiol; Estrogen Antagonists; Ethanol; Female; Gestonorone Caproate; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Middle Aged; Neoplasm Metastasis; Neoplastic Stem Cells; Ovarian Neoplasms; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen

The Ca2+- and phospholipid-dependent phosphotransferase activity of protein kinase C was inhibited by the triphenylethylene compounds clomiphene [drug concentration causing 50% inhibition (IC50) = 25 microM], 4-hydroxytamoxifen (IC50 = 25 microM), and N-desmethyltamoxifen (IC50 = 8 microM). The Ca2+- and phospholipid-independent phosphorylation of protamine sulfate, which is catalyzed by protein kinase C, was not inhibited by the triphenylethylenes, suggesting that they do not interact directly with the active site of protein kinase C. The inhibitory potency of each triphenylethylene was reduced when the phospholipid concentration was increased, providing evidence that these drugs inhibited protein kinase C by interacting with phospholipids. The potencies of the effects of the triphenylethylenes on protein kinase C in the lipid environment of intact cells were evaluated by determining their efficacies in the inhibition of [3H]phorbol 12,13-dibutyrate (PDBu) binding to mouse embryo C3H/10T1/2 cells. Micromolar concentrations of each drug inhibited [3H]PDBu binding in these cells. N-Desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen inhibited protein kinase C with the same order of potency as that which has been reported for their inhibition of MCF-7 cell growth by Reddel et al. (1983). N-Desmethyltamoxifen and 4-hydroxytamoxifen were also more potent than tamoxifen in the inhibition of the growth of mouse embryo fibroblast C3H/10T1/2 cells. These correlations suggest that the mechanism of growth inhibition by tamoxifen and its metabolites includes interactions with protein kinase C.

Topics: Animals; Brain; Cells, Cultured; Estrogen Antagonists; Fibroblasts; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Rats; Stilbenes; Structure-Activity Relationship; Tamoxifen

Methods and evaluation of the human tumor stem cell assay (HTSCA) are described. Advantages and disadvantages of the test system are elaborated. The in vitro/in vivo correlation in the drug screening of human ovarian carcinomas shows that the prediction of sensitivity to a cytotoxic agent is only possible in 64%. Prediction of drug resistance, however, seems to be possible in 95%. The number of patients that profit from the HTSCA seems to be only less than 10%. Our investigations describe the influence of various hormones and antiestrogens on the colony formation of human ovarian carcinoma cells. Tamoxifen and his major metabolite 4-hydroxy-tamoxifen were the most active agents. Both compounds inhibit the colony survival (70% at pharmacological concentrations) of 60% of the screened ovarian carcinomas. A significant correlation to the quantitative level of estrogen or progesterone receptors could not be proved. Colony formation of ovarian carcinoma cells was compared in the HTSCA as described by Hamburger and Salmon and in a methylcellulose-monolayer system. Our results show that the colony formation corresponds to the results of the original HTSCA: Cloning ovarian carcinoma cells in the methylcellulose-monolayer, however, seems to be technically easier and faster.

Topics: Agar; Antineoplastic Agents; Carcinoma; Cells, Cultured; Colony-Forming Units Assay; Drug Resistance; Estradiol; Female; Hormones; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Methylcellulose; Ovarian Neoplasms; Progesterone; Prognosis; Tamoxifen; Tumor Stem Cell Assay

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

Tamoxifen (Nolvadex; TAM) and its major metabolites, N-desmethyl- (DMT) and 4-hydroxy-tamoxifen (HT), were shown to be potent inhibitors of hepatic cytochrome P-450-dependent mixed function oxidations. From in vitro experiments, all three were found to be potent inhibitors of oxidation of Type-I substrates (ethylmorphine and aminopyrine) and less potent, non-competitive inhibitors of Type-II substrates (aniline and dimethylnitrosamine). TAM, DMT and HT were of essentially equal potency and had a much more pronounced effect on Type-I substrates than on Type-II compounds studied. Their action appears to parallel SKF-525A in type and potency of inhibition produced. Spectral binding studies suggest that TAM and its metabolites exert their effects by occupying the Type-I binding site of cytochrome P-450 and thus limiting the accessibility of other substrates to the active site of the enzyme. TAM (and its metabolites) also inhibits its own metabolism, altering the distribution and elimination half-lives of tamoxifen-derived species. In addition, tamoxifen metabolism was found to be sensitive to the presence of other drugs. These results raise concern regarding the role that continued administration of tamoxifen plays in changing its own disposition as well as in the detoxification of drugs administered with it.

Topics: Aminopyrine N-Demethylase; Aniline Hydroxylase; Animals; Ethylmorphine-N-Demethylase; Female; Kinetics; Microsomes, Liver; Rats; Rats, Inbred Strains; 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

Sensitive and reproducible analyses were developed for assaying tamoxifen, monohydroxytamoxifen, N-desmethyltamoxifen, metabolite E [trans-1(4-hydroxyphenyl)1,2-diphenylbut-1-ene] and a new metabolite, metabolite Y [trans-1(4-hydroxyethoxyphenyl)-1,2-diphenylbut-1-ene] in human serum using high-performance liquid chromatography (HPLC). Three different systems were developed for specific purposes. All chromatography was performed using serum extracts made with hexane-butanol. Detection was by fluorimetry of phenanthrene derivatives formed by on-stream UV irradiation with a newly described device for post-column irradiation of the HPLC stream. This device may be of use in other HPLC systems requiring post-column photochemical reactions.

Topics: Animals; Chromatography, High Pressure Liquid; Dogs; Fluorometry; Humans; Tamoxifen

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