Accelerator mass spectrometry allows for cellular quantification of doxorubicin at femtomolar concentrations.

Accelerator mass spectrometry (AMS) is a highly sensitive analytical methodology used to quantify the content of radioisotopes, such as (14)C, in a sample. The primary goals of this work were to demonstrate the utility of AMS in determining total cellular [(14)C]anthracycline concentrations following administration of doxorubicin (DOX) and to develop a sensitive assay that is superior to high performance liquid chromatography (HPLC) for the quantification of [(14)C]anthracycline at the tumor level. In order to validate the sensitivity of AMS versus HPLC with fluorescence detection, we performed three studies comparing the cellular accumulation of DOX: one in vitro cell line study, and two in vivo xenograft mouse studies. Using AMS, we quantified cellular [(14)C]anthracycline content up to 4 h following in vitro exposure at concentrations ranging from 0.2 pg/ml (345 fM) to 2 microg/ml (3.45 microM) [(14)C]DOX. The results of this study show that, compared to standard fluorescence-based HPLC, the AMS method was over five orders of magnitude more sensitive. Two in vivo studies compared the sensitivity of AMS to HPLC using a nude mouse xenograft model in which breast cancer cells were implanted subcutaneously. After sufficiently large tumors formed, [(14)C]DOX was administered intravenously at two dose levels. Additionally, we tested the AMS method in a nude mouse xenograft model of multidrug resistance (MDR) in which each mouse was implanted with both wild type and MDR+ cells on opposite flanks. The results of the second and third studies showed that [(14)C]anthracycline concentrations were significantly higher in the wild type tumors compared to the MDR+ tumors, consistent with the MDR model. Although this method does not discriminate between parent drug and metabolites, the extreme sensitivity of AMS should facilitate similar studies in humans to establish target site drug delivery and to potentially determine the optimal treatment dose and regimen.

Effects of ospemifene (FC-1271a) on uterine endometrium, vaginal maturation index, and hormonal status in healthy postmenopausal women.

OBJECTIVE: Selective estrogen receptor modulators (SERMs) are drugs that exhibit both estrogen agonistic and antagonistic effects that are tissue-specific. Ospemifene (FC-1271a) is a novel SERM compound, which has been shown in animal models to have estrogen-like effects on bone and the cardiovascular system, while having antiestrogen-like effects in uterus and breast. In this study, we investigated the effects of ospemifene on the uterine endometrium, vaginal maturation index and hormonal status in healthy postmenopausal women. METHODS: The study was conducted as a double-blind, placebo-controlled phase I study, where 40 healthy postmenopausal women volunteers were randomized to receive daily oral doses of ospemifene either 25, 50, 100 or 200 mg or placebo for 12 weeks. Vaginal ultrasonography and endometrial biopsy were performed and vaginal maturation index determined at baseline and at 12 weeks' visit. Serum concentrations of estradiol, luteinizing hormone, follicle stimulating hormone (FSH), sex-hormone binding globulin (SHBG), parathyroid hormone and prolactin were determined from samples taken at baseline, at 4 days and at 4, 12, and 16 weeks' visits. Climacteric symptoms were assessed using 12 visual analogue scales (VAS) at baseline and at the end of the study. RESULTS: No clinically significant changes were seen in endometrial thickness at any dose level. Ospemifene exerted a very weak estrogenic effect on endometrial histology. On the other hand, it induced a clear estrogenic effect on vaginal epithelium. Among the endocrine parameters only FSH and SHBG showed significant dose dependent changes; FSH decreased and SHBG increased during the treatment. In general, ospemifene was well tolerated. The 25 and 50 mg doses tended to reduce climacteric symptoms, but no statistically significant differences were observed between different doses of ospemifene and placebo. The highest dose level (200 mg) induced more subjective adverse reactions, especially hot flushes, than lower doses. CONCLUSION: Our study suggests that a safe and well tolerated dose of ospemifene for potential clinical use may be between 25 and 100 mg. Further studies are needed to substantiate the results of this Phase I pilot study.

High-dose tamoxifen added to concurrent biochemotherapy with decrescendo interleukin-2 in patients with metastatic melanoma.

BACKGROUND: In vitro cell culture data and preclinical models suggest that tamoxifen modulates tumor cell sensitivity to a wide range of therapeutic agents. In the current study, the authors examined whether high-dose tamoxifen (HDT) improved the overall and complete response in patients with metastatic melanoma who were treated with concurrent biochemotherapy. METHODS: Forty-nine patients were treated with a biochemotherapy regimen of dacarbazine, vinblastine, cisplatin, decrescendo interleukin-2, interferon-alpha-2b, and tamoxifen. The study had a 2-step design, beginning with a tamoxifen dose escalation from 40 mg to 320 mg (17 subjects) to evaluate safety and tolerability, followed by Phase II accrual of 32 patients to HDT (320 mg) to assess clinical efficacy. Efficacy was compared with a similar modified biochemotherapy regimen with low-dose tamoxifen (LDT). Pharmacokinetic studies were performed to determine in vivo tamoxifen levels. RESULTS: Tamoxifen dose escalation was completed without any reported dose-limiting toxicity. The overall response rate in the HDT group was 50% (95% confidence interval, 33.2%-66.8%), with a complete response rate of 6% and a median survival of 9.5 months. The overall response rate was not improved and the complete response and survival appeared inferior compared with that of patients recently treated with concurrent biochemotherapy and LDT. Serum tamoxifen levels were found to correlate with the dose administered, with a mean of 0.9 microM at the 40-mg dose to 4.6 microM at the 320-mg dose. Ultrafiltered protein-free sera demonstrated low (< 0.01 microM) concentrations of tamoxifen. CONCLUSIONS: The addition of HDT to a regimen of concurrent biochemotherapy did not appear to improve response rates or overall survival, despite reaching the targeted plasma concentration. Unknown drug interactions or high protein binding of tamoxifen may account for the lack of clinical effectiveness.

In vitro and in vivo biologic effects of Ospemifene (FC-1271a) in breast cancer.

Ospemifene (FC-1271a) is a novel selective estrogen receptor modulator under development for osteoporosis prevention. In the present paper, we examine both the in vitro and in vivo effects of FC-1271a in breast cancer models. In vitro, the growth inhibitory effects of FC-1271a and its main metabolite are investigated in MCF-7 and MDA-MB-231 human breast cancer cells at doses ranging from 0.1 to 10 microM. Modulation of pS2 expression, an indicator of estrogen activity, was also examined in all experiments using reverse transcription-polymerase chain reaction. In vivo, the effects of treatment with 10, 25, 50, or 100 mg/kg FC-1271a on MCF-7 and MDA-MB-231 human tumor xenografts in athymic, ovariectomized mice were determined. For MCF-7 cells, FC-1271a and its main metabolite, toremifene VI (TOR VI) displayed anti-estrogenic effects in vitro as shown through growth inhibition and decreased expression of pS2. Treatment with FC-1271a in vivo inhibited MCF-7 tumor growth, compared with control (P< or =0.05). FC-1271a and TOR VI did not inhibit the growth of MDA-MB-231 cells in vitro, and no clear effects of FC-1271a treatment were seen on MDA-MB-231 tumor growth in vivo. In conclusion, FC-1271a appears to exert anti-estrogenic effects dependent on estrogen receptor positivity in vitro and in vivo on the growth of MCF-7 cells.

Clinical pharmacokinetics of toremifene.

Toremifene is a chlorinated triphenylethylene derivative of tamoxifen approved for use in the treatment of patients with metastatic breast cancer. Toremifene is well tolerated in patients, and common adverse effects of this drug include vasomotor symptoms such as hot flashes and vaginal discharge. This compound is administered to patients orally at a dose of 60 mg/day, although alternative methods of administration have been investigated. Oral bioavailability is estimated to be approximately 100%. At steady state, toremifene and its metabolites are highly protein bound (>95%). Toremifene is metabolised in the liver by cytochrome P450 enzymes, and it is eliminated primarily in the faeces following enterohepatic circulation. The half-life of toremifene is approximately 5 days, and steady state is reached by 6 weeks depending on the dose given. The pharmacokinetics of toremifene have been shown to be altered by certain liver conditions, but age and kidney function do not appear to be as significant.

Pharmacokinetics of (deaminohydroxy)toremifene in humans: a new, selective estrogen-receptor modulator.

PURPOSE: New selective estrogen-receptor modulators for the treatment and prevention of osteoporosis, cardiovascular disease and breast cancer are currently the focus of intense research. (Deaminohydroxy)toremifene (Z-2-[4-(4-chloro- 1,2-diphenyl-but-1-enyl)phenoxy]ethanol; FC-1271a) has been shown to prevent bone resorption in rats while having no or weak estrogen-like effects on the uterus, which makes it a good candidate drug for osteoporosis prevention. Our purpose here was to examine the pharmacokinetics of (deaminohydroxy)toremifene in humans included in two phase-I studies. METHODS: The first was a single-dose, dose-escalation study with 28 healthy male volunteers. Doses ranged from 10 mg to 800 mg. The second study was conducted during a 12-week period with 40 healthy, post-menopausal women, who received repeated oral doses of 25-200 mg. Standard pharmacokinetic parameters were assessed. RESULTS: In the single-dose study, time to reach peak concentration (tmax) ranged from 1.3 h to 4.0 h; peak concentration (Cmax) ranged from 15 ng/ml to 445 ng/ ml; and the estimated terminal elimination half-life (mean +/- SD; t1/2) was 24.8 +/- 7.0 h. In the repeated-dose study, tmax ranged from 1.9 h to 2.6 h at 6 weeks and from 2.5 h to 2.9 h at 12 weeks. Cmax ranged from 295 ng/ml to 1,043 ng/ml at 6 weeks and from 25 ng/ml to 1211 ng/ml at 12 weeks. The average t1/2 at all dose levels was 29.7 +/- 1.5 h (overall mean +/- SD). Strong linear correlations between the dose and Cmax and between the dose and the area under the curve were observed in both studies. CONCLUSION: Our results indicate that (deaminohydroxy)toremifene has pharmacokinetics suitable for single daily dosing. The prophylactic use of this agent in women susceptible to development of osteoporosis, cardiovascular disease and breast cancer could, therefore, be tested using a once-daily dosing schedule similar to those of other hormone-replacement therapy regimens.

Genotoxic effects of the novel mixed antiestrogen FC-1271a in comparison to tamoxifen and toremifene.

Tamoxifen has been used for the treatment of breast cancer since the 1970s, but is considered a carcinogen because it has been linked to liver cancer in rats and an increased risk of endometrial cancer in patients. In rats, DNA adducts appear to be responsible for carcinogenesis, but their contribution to carcinogenesis in humans is not clear. FC-1271a and toremifene are mixed antiestrogens similar to tamoxifen. In order to compare the genotoxicity of these different triphenylethylenes, we treated mice for 28 days with 50 mg/kg of either tamoxifen, toremifene, FC- 1271 a or vehicle control. DNA from liver and uterus was assayed by standard 32P-postlabeling and thin layer chromatography for the presence of DNA adducts. Two methods of drug administration (oral and subcutaneous) and two strains of mice were compared and the plasma and tissue concentrations of the drugs and three metabolites of tamoxifen and toremifene were determined. Regardless of the conditions, only tamoxifen-treated mice showed DNA adducts in the liver. Adduct levels did not correlate with drug or metabolite levels and adducts were present even when drug was not detectable. Mice were also treated orally with either 50, 100, or 200 mg/kg of drug for 7 days. Again, adducts were found only in liver tissue of mice treated with tamoxifen, and adduct levels were dose-dependent. In conclusion, the chlorinated triphenylethylene FC-1271a did not cause DNA adducts under various conditions in mice, suggesting a low carcinogenic potential.

Quantitative analysis of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol in human plasma using high-performance liquid chromatography.

A highly sensitive and precise high-performance liquid chromatography (HPLC) assay was developed and validated for the quantitation of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl) phenoxy]ethanol (FC-1271a) in human plasma. Plasma samples (1.0 ml) containing FC-1271a and internal standard (toremifene citrate; Farestono) were extracted using a 2% 1-butanol, 98% hexane solution with an extraction efficiency of >97%. Samples were reconstituted in methanol, irradiated with high intensity ultraviolet light (254 nm) for 1 min, and injected onto a C18 reverse phase column. Samples were eluted isocratically at a flow-rate of 0.5 ml/min with a mobile phase consisting of 6.5% water and 0.5% triethylamine in methanol. The fluorescence of photochemically activated compounds was detected using a fluorometer set at an excitation wavelength of 266 nm and emission wavelength of 370 nm. Under these assay conditions, standard calibration curves were linear through a concentration range of 10-400 ng/ml. In summary, we have developed and validated an HPLC assay to quantitate FC-1271a in human plasma.

Hormone replacement therapy and breast cancer: revisiting the issues.

OBJECTIVE: To assess current ideas about the benefits and risks of estrogen and hormone replacement therapy (ERT/HRT) in postmenopausal women. DATA SOURCES: MEDLINE searches, supplemented by various texts, of the literature on HRT, ERT, and selective estrogen receptor modulators (SERMs): tamoxifen, toremifene, and raloxifene. DATA SYNTHESIS: HRT is primarily used for improving quality of life in women suffering from vasomotor symptoms associated with menopause. HRT is beneficial in postmenopausal women for preventing cardiovascular disease, osteoporosis, and Alzheimer's disease. Review of meta-analyses of clinical trials showed that ERT/HRT ever-users (patients who have ever used ERT/HRT) did not have an increased risk of breast cancer, but current users did have an increased risk, with some studies reporting increasing risk with duration of ERT. No relationship was found between dose or the addition of progestin to ERT and increased breast cancer risk. Overall breast cancer mortality rates associated with HRT were decreased in current users. In general, HRT does not increase the risk of breast cancer in women with a family history of the disease, compared with those without a family history. New HRT strategies that could potentially prevent breast cancer are now being developed. The SERMs tamoxifen and toremifene appear to have positive clinical effects on bone and serum lipids; they are currently being investigated for use as breast cancer chemopreventive agents. Raloxifene, a new SERM used for the prevention of osteoporosis, is an alternative for women who cannot tolerate HRT. Unfortunately, these SERMS have anti-estrogenic effects and thus cause vasomotor adverse effects such as hot flashes and vaginal dryness. In addition, SERMs do not protect against heart disease or prevent osteoporosis as well as does HRT. CONCLUSION: Presently, SERMs will not become first-line HRT, as the positive effects of ERT/HRT may outweigh any potentially increased risk of breast cancer. The development of new agents with pharmacodynamic profiles similar to that of ERT/HRT but lacking its adverse effects would be greatly beneficial for postmenopausal women.

Intrinsic reactivity of tamoxifen and toremifene metabolites with DNA.

The antiestrogen tamoxifen is known to cause liver cancer in rats. This may be due to the formation of abundant DNA adducts in rat liver. A likely precursor to some of the tamoxifen adducts in rats is alpha-hydroxytamoxifen. It is not clear whether the rat data are relevant to human exposure. In the present study, we show that one of the major metabolites in humans reacts with double-stranded DNA in vitro in the absence of any metabolizing enzymes or activating chemicals. At least two distinct adduct spots resulting from 4-hydroxy-N-desmethyltamoxifen (metabolite Bx) were detected by 32P postlabeling and thin layer chromatography. The adduct level increases dramatically when metabolite Bx is irradiated with UV light to fuse into a phenanthrene ring system. 4-hydroxy-N-desmethyltoremifene, which differs from Bx by a single chlorine atom, forms fewer DNA adducts without irradiation but similar amounts after irradiation. These results suggest that the chlorine atom may interfere with drug-DNA interactions which facilitate adduct formation.

Pharmacokinetic analysis of high-dose toremifene in combination with doxorubicin.

PURPOSE: Toremifene (Fareston) is an orally administered triphenylethylene derivative with chemosensitizing activity in vitro in estrogen receptor-negative multidrug-resistant human breast cancer cells. The purpose of this study was to evaluate the effects of high-dose toremifene (600 mg/day for 5 days) on the plasma pharmacokinetics of doxorubicin in humans. The 600-mg dose had been previously established as the maximum tolerated dose in a phase I study of 35 patients. METHODS: Doxorubicin was administered as an intravenous (i.v.) bolus over 15 min at a dose of 60 mg/m2 to 11 patients in the absence of toremifene pretreatment to establish baseline doxorubicin pharmacokinetics. Six of these patients received 600 mg/day toremifene for 5 days 4 weeks later, followed by an i.v. bolus dose of doxorubicin (60 mg/m2) on day 5. During toremifene pre-treatment, blood specimens (5 ml) were drawn at 0, 2, 4, and 24 h after dosing to assess peak levels. Following doxorubicin administration in each cycle, blood specimens were collected over a 72-h period for determination of the terminal half-life of elimination. Plasma concentrations of doxorubicin and toremifene were assessed by high-performance liquid chromatography (HPLC). Cumulative linear areas under the time-concentration curve (AUC) for doxorubicin were calculated using a noncompartmental model. RESULTS: Prior to toremifene dosing, baseline doxorubicin pharmacokinetic studies showed an average terminal half-life of elimination of 40.04+/-7.86 h in 4 patients, and an average AUC of 135 600+/-67 600 microg/ml.h in 11 patients. In 4 of the patients receiving 600 mg/day toremifene for 5 days, the average terminal half-life of elimination was 38.12+/-7.81 h, and the average AUC was 141 900+/-62 900 microg/ml.h in 6 patients, i.e. a slight increase of 4.6%. No statistically significant change in the doxorubicin elimination kinetics with or without toremifene therapy was observed. CONCLUSIONS: Toremifene does not appear to interfere with the elimination kinetics of doxorubicin.

Intratumoral toremifene therapy and tissue distribution in the baboon.

The purpose of the present study was to evaluate the tissue distribution of toremifene (TOR) in baboons following intra-tissue injections and to examine the effectiveness of intratumoral TOR therapy of baboons with various spontaneous neoplasms. Five healthy baboons (Papio sp.) were used to examine the distribution of TOR following intra-tissue injections. Twenty-three different tissue specimens were collected for HPLC analysis. In addition, four baboons with various spontaneous neoplasms (myxoma, squamous cell carcinoma, lymphosarcoma and adenocarcinoma) were treated with intratumoral TOR and their responses were evaluated. Tissue TOR distribution was also examined in these animals. In the tissue distribution study, target tissue/serum TOR concentration ratios ranged from 138 to 8873 and the target tissue/other tissue ratios ranged from 1.2 to 2428. The distribution of TOR was very favorable, with the highest concentrations outside the injection sites noted in adjacent organs. A marked response was observed in the myxoma and partial responses were observed in the other three cases. Drug level analysis data from these four animals revealed tissue concentrations similar to those seen in the TOR tissue distribution study. Intratumoral administration of TOR can achieve effective local tumor and tissue concentrations, while systemic distribution via circulation to other organs is limited.

Tissue distribution of transdermal toremifene.

PURPOSE: Toremifene is an orally administered triphenylethylene derivative with antiestrogenic activity that is primarily used in the treatment of patients with metastatic breast cancer. The purpose of this study was to evaluate the therapeutic advantage of local (transdermal) administration of toremifene in several animal models. Local (subcutaneous and skin) versus systemic concentrations of toremifene were evaluated serially following transdermal application of the drug. With high local concentrations and minimal distribution to other organs via the circulation, topical toremifene may deliver maximal therapeutic effects to local tissue while avoiding the side effects seen with systemic therapy. METHODS: Three animal models (nude mice, baboons, and a horse) were used to examine topically administered toremifene for kinetic measurements. RESULTS: In nude mice implanted subcutaneously with MDA-MB-231 human breast tumors, topical toremifene (2.5 mg/day x 5 days) produced greater than 50-fold higher tumor concentrations compared with intraperitoneal (i.p.) administration (1.0 mg/day x 5 days). Systemic distribution in plasma, uterus, and liver was lower following topical than following i.p. administration. In nude mice inoculated subcutaneously with estrogen receptor-positive (ER +) MCF-7 human breast cancer cells, topical toremifene and 4-hydroxytoremifene (4-OH) prevented tumor growth in the presence of estradiol. In four nontumor-bearing baboons that were given transdermal toremifene, relatively high distribution of drug was noted in normal breast tissue and fat, compared with undetectable serum concentrations. Finally, a new topical formulation of toremifene (a gel preparation for human use, Orion-Farmos, Finland) achieved high local tumor toremifene concentrations in a horse melanoma, with minimal systemic distribution. CONCLUSIONS: Transdermal toremifene can achieve high local tissue concentrations with minimal systemic distribution.

Haloenol lactone: a new synergist of chemotherapy in vitro.

Over-expression of glutathione S-transferases (GST) has been found to play a significant role in multiple drug resistance in cancer chemotherapy. To combat GST-mediated drug resistance, GST inhibitors are being studied as potential synergists for effective cancer chemotherapy. We have designed and synthesized a haloenol lactone derivative as a mechanism-based inactivator of GST-pi isozyme. In the current study, we examined the inhibitory effect of the haloenol lactone compound on GST of a human renal carcinoma cell line UOK130 and found that this compound shows time-dependent GST inhibition in these cancer cells. The enzyme activity lost upon incubation with the haloenol lactone could not be restored by extensive dialysis against buffer. Pretreatment of the cancer cells with 1.0 microM of haloenol lactone increased cytotoxicity induced by cisplatin in the UOK130 cell line. This report further supports the possibility of synergizing alkylating agents in cancer chemotherapy by use of selective GST inhibitors.

Tamoxifen for the prevention of breast cancer: no.

The tamoxifen chemoprevention trial in health women is ongoing in over 250 cancer treatment centers. The use of tamoxifen for the treatment of postmenopausal women with known breast cancer has been touted as a medical breakthrough by many physicians. However, the ongoing trial which enroll high-risk healthy women above the age of 34, has been controversial since its initiation in 1991. Congressional hearings, editorial, and statistical analyses concerning the scientific basis of the trial have emerged over the past year. This chapter presents the newest scientific facts, and speculates on the unknown risks of using tamoxifen as a chemopreventive in healthy women.

Flow cytometry: potential utility in monitoring drug effects in breast cancer.

Flow cytometric analysis of DNA ploidy and S-phase fraction are well recognized prognostic indicators in breast cancer. The present paper deals with the widening of the applications of flow cytometry to monitoring the effectiveness of antiestrogen therapy, detecting clonal selection and emergence of drug resistance, and monitoring chemosensitizing properties of drugs. Antiestrogen activity can be studied by DNA flow cytometry to address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance. Patient plasma specimens containing various concentrations of triphenylethylenes can be monitored for drug-induced effects using cell cycle measurements and correlated to in vivo drug levels. DNA flow cytometry has also been instrumental in the study of the effects of prolonged low-dose (0.5 microM for > 100 days) tamoxifen treatment on human estrogen receptor negative MDA-MB-231 cells, where it was shown that tamoxifen may significantly alter cell cycle kinetics and tumorigenicity of these cells, selecting a new, more aggressive, and rapidly growing clone. Lastly, it has been shown that the chemosensitizing properties of another triphenylethylene antiestrogen, toremifene, on estrogen receptor negative, multidrug resistant MDA-MB-231-A1 human breast cancer cells can be studied using flow cytometric analysis. Toremifene (and its metabolites N-desmethyltoremifene and toremifene IV) are able to "resensitize" MDA-MB-231-A1 cells to vinblastine and doxorubicin, as reflected in a marked shift of cells to G2/M phase of the cell cycle. Flow cytometry is a widely available technique that might be applied clinically to monitor, at the cellular level, drug effects on tumors, including the modulators of drug resistance.

Tamoxifen resistance in breast cancer.

Tamoxifen (TAM) resistance is the underlying cause of treatment failure in many breast cancer patients receiving TAM. The mechanism(s) involved in TAM resistance are poorly understood. A variety of mechanisms have been proposed but only limited evidence exists to substantiate them. Studies have now shown that in many patients TAM resistance is not related to the down regulation or loss of estrogen receptors (ER). Variant ER have been identified, but their significance clinically remains to be proven. Since breast cancer cells secrete several estrogen-regulated growth factors and growth inhibitors that may have autocrine or paracrine activity, altered growth factor production is another possible mechanism for TAM resistance. Tissue-specific transcription activating factors that may alter how the signal induced by TAM binding to the receptor is interpreted by the cell also require further investigation. An increase in antiestrogen binding sites (AEBS), which could effectively partition TAM and reduce its concentration at the ER has also been proposed as a potential mechanism. Pharmacologic mechanisms, such as a shift in metabolism toward the accumulation of estrogenic metabolites, are supported by recent data demonstrating metabolite E and bisphenol in tumors from TAM-resistant patients. Furthermore, a decrease in tumor TAM accumulation and an altered metabolite profile have been reported in TAM-resistant breast tumors grown in nude mice. These and other studies suggest that TAM resistance may be multifactorial in nature, but definitive identification of mechanisms that are operative in clinical TAM resistance requires further study.

A breast cancer clone selected by tamoxifen has increased growth rate and reduced sensitivity to doxorubicin.

The in vivo growth rate and the chemosensitivity patterns of a cell clone selected by tamoxifen from the estrogen receptor-negative human breast cancer cell line MDA-MB-231 was studied in the nude mouse model and with flow cytometry. To investigate the growth rate of the wild-type and clone cells in vivo, the cells were inoculated into the opposite flanks of 5 male nude mice. Drug sensitivity to doxorubicin (10 ng/mL), vinblastine (1 ng/mL), and paclitaxel (1 ng/mL) was examined in wild-type/clone cell mixture using flow cytometry. Northern blot technique was used to study the expression of mdr-1 messenger RNA in both the wild-type and the clone cells. The tumors derived from the clone and wild-type cells were, following a 3-week growth period, 260.2 +/- 78.8 mm2 vs. 68.3 +/- 50.8 mm2 in size, respectively (P < 0.001). Following a 28-day continuous exposure, doxorubicin was selectively, toxic to the wild-type cells, while having no apparent effect on the clone population. However, paclitaxel- and vinblastine-treated wild-type/clone cell mixtures did not exhibit a differential cytotoxic effect on either cell population. It was concluded that the clone selected by tamoxifen shows an aggressive growth rate in vivo and an altered chemosensitivity pattern to doxorubicin in vitro.