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.

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo.

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.

Toremifene enhances cell cycle block and growth inhibition by vinblastine in multidrug resistant human breast cancer cells.

The clinical study of compounds that modulate multidrug resistance has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at the level of the tumor cell. Previously, toremifene has been shown to be well tolerated clinically and to sensitize multidrug resistant cells to the effects of cytotoxic chemotherapeutic agents. The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-MB-A1 human breast cancer cells were studied using flow cytometric analysis and growth inhibition assays. Cell cycle kinetics of MDA-MB-A1 cells were not significantly affected by treatment with either toremifene, N-desmethyltoremifene, Toremifene IV or vinblastine alone, as the majority of cells remained in G0/G1. However, preincubation with toremifene or one of its metabolites for 72 hours followed by treatment for one hour with vinblastine caused a marked shift of cells to G2/M, as cells appeared to be blocked in that phase of the cell cycle. This result was nearly identical to the effect of vinblastine alone on vinblastine-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-MB-A1 cells to vinblastine. This chemosensitizing effect of toremifene was accompanied by an enhanced inhibition of cell growth by vinblastine. The chemosensitizing effects of toremifene or one of its metabolites in combination with cytotoxic chemotherapy can be effectively monitored by flow cytometry, an easily accessible technique.

Targeting chemosensitizing doses of toremifene based on protein binding.

Toremifene is currently being evaluated as a chemosensitizing agent in doxorubicin-resistant patients. Although concentrations of > 2 microM reverse resistance in vitro, target concentrations required to reverse multidrug resistance (MDR) in vivo may be highly influenced by variables such as protein binding in serum. We examined the effects of high serum concentrations on the cellular accumulation of toremifene in an MDR MDA-MB-A-1 human breast-cancer cell line. We then examined the cellular accumulation of doxorubicin at various toremifene concentrations in 5% - 100% serum. We also measured the concentrations of toremifene and its major metabolites in plasma specimens obtained from two patients receiving 360 mg/day for 5 days in a phase I study. Our results show that (1) high serum concentrations decrease toremifene accumulation, (2) toremifene concentrations of < or = 2.5 microM enhance doxorubicin accumulation, and (3) patients achieve plasma toremifene concentrations of 10-15 microM following doses of 360 mg/day x 5 days. Our findings suggest that in vivo toremifene concentrations well above those used to reverse resistance in vitro are required to overcome the effect of high serum-protein binding.

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.

Monitoring the chemosensitizing effects of toremifene with flow cytometry in estrogen receptor negative multidrug resistant human breast cancer cells.

The clinical study of compounds that modulate multidrug resistance in cancer cells has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at a cellular level. The non-steroidal triphenylethylene toremifene is well tolerated clinically and can sensitize multidrug resistant cells to the effects of doxorubicin in vitro. The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-A1 human breast cancer cells were studied using flow cytometric analysis. Cell cycle kinetics of MDA-A1 cells were not significantly affected by treatment with either toremifene or doxorubicin alone, as the majority of cells remained in G0/G1. However, preincubation with toremifene for 70 hours followed by treatment with doxorubicin caused a marked shift of cells to G2, as cells appeared to be blocked in that phase of the cell cycle. This result was nearly identical to the effect of doxorubicin alone on doxorubicin-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-A1 cells to doxorubicin. This chemosensitizing effect of toremifene was accompanied by an enhanced accumulation of doxorubicin in MDA-A1 cells (+110% after 70 hours pre-incubation with toremifene), and by a depression in protein kinase C activity in MDA-A1 cells that was maximal following 70 hours incubation with toremifene. Flow cytometry is a widely available technique that might be applied clinically to monitor at the cellular level the chemosensitizing effects of toremifene and other modulators of multidrug resistance.