Toremifene Research Articles

High-dose toremifene as first-line treatment of metastatic breast cancer resistant to adjuvant aromatase inhibitor: A multicenter phase II study

There is currently no standardized therapy available for metastatic breast cancer in patients with aromatase inhibitor (AI)-resistant breast cancer. We conducted a prospective study to examine the efficacy and safety of high-dose toremifene (TOR) treatment for the first-line treatment of metastatic breast cancer following AI adjuvant therapy. A multicenter phase II study was designed (Registry no.: UMIN000000489). Inclusion criteria comprised hormone-responsive postmenopausal women who had received adjuvant AI postoperatively for >1 year and had relapsed during the treatment or within 12 months of completion of adjuvant therapy. Treatment comprised oral intake of 120 mg TOR once a day. The primary endpoint was objective response rate (ORR). The secondary endpoints were evaluations of clinical benefit (CB), progression-free survival (PFS) and toxicity. A total of 13 patients were enrolled. ORR was 7.7% (1/13) [95% CI, 0.2-36.0%]. In total, 7 patients (53.8%) had stable disease (SD), 5 of whom were long SD, and 5 patients (38.5%) experienced progressive disease (PD). The CB rate was 46.2% (6/13) [95% CI, 19.2-74.9%]. The median time to PFS was 5.9 months. No serious adverse events were observed. Patients with HER2-positive disease exhibited marginally poorer PFS (p=0.08). Patients with PD had a relatively short duration of AI treatment in contrast to responders, who had a longer period of AI treatment (p=0.02). High-dose TOR as a first-line treatment following AI adjuvant therapy was effective and well tolerated. A longer duration of adjuvant AI therapy and negative HER2 overexpression may, with further studies, be beneficial as positive predictive factors for the effectiveness of TOR treatment.

Novel strategy to restore the activity of taxanes: a multicenter phase II study of combination therapy with taxanes and toremifene at 120 mg for advanced/recurrent breast cancer. (Kinki Multidisciplinary Breast Oncology Group: KMBOG0612)

Background: A preclinical study reported that toremifene (TOR) increased the level of an anticancer agent such as doxorubicin and paclitaxel in the breast cancer cell which has acquired resistance to them by the expression of P-glycoprotein.

Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells

To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene. Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines. Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake. Taken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.

Effects of toremifene and anastrozole on serum lipids and bone metabolism in postmenopausal females with estrogen receptor–positive breast cancer: the results of a 2-year multicenter open randomized study

The potential long-term adverse effects on quality of life have to be considered when selecting agents for adjuvant hormonal treatment for postmenopausal patients with estrogen receptor-positive breast cancer. We performed a 2-year multicenter randomized study to assess the differences in the time course effects between toremifene (TOR) and anastrozole (ANA) on serum lipid profiles and bone metabolism. This study assessed the serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A-1 (Apo A1), and apolipoprotein B (Apo B) as lipid profiles and bone-specific alkaline phosphatase (BAP) and the N-telopeptide of type-I collagen (NTX) as bone turnover markers in patients who received daily doses of 40 mg and 1 mg for TOR and ANA, respectively. A decreased serum level of TC, LDL-C, and Apo B was, respectively, observed at 6 months in 6.2, 12.9, and 13.8% of the patients who received TOR compared with the baseline. These decreases were maintained for at least 24 months. These lipid levels were not changed in those who received ANA. In the TOR patients, there was an increase in the serum level of HDL-C and Apo A1 at 6 months in 17.1 and 16.3%, respectively, which was maintained for at least 24 months, whereas these levels were almost stable in the patients who received ANA. Serum BAP decreased by 12.1% at 12 months and further decreased at 24 months and the serum NTX decreased by 22.0% at 6 months, which was maintained for at least 24 months in the patients who received TOR. In contrast, the serum BAP was increased by 26.0% at 6 months and by 29.2% at 12 months and the serum NTX increased by 21.3% at 24 months compared with baseline in those received ANA. However, the serum BAP increase was not significant at 24 months. TOR provides better effects than ANA in terms of lipid profiles and bone metabolism in postmenopausal females with early breast cancer.

Importance of CYP2D6 metabolism in the in vitro antiestrogenic activity of toremifene and tamoxifen.

e13067 Background: Toremifene (TOR) and tamoxifen (TAM) are approved treatments for advanced breast cancer. It has been suggested that poor metabolizers of TAM have worse clinical outcomes than patients who exhibit normal TAM metabolism. TOR and TAM are extensively metabolized by the human cytochrome P450 system (TAM - 3A4 and 2D6; TOR - 3A4). To gain a better understanding of CYP2D6 status and the potential impact on breast cancer activity, we investigated the estrogen and antiestrogen actions of TOR and TAM and their NDM, 4-OH, and 4-OH-NDM metabolites in Ishikawa endometrial and MCF-7 breast cancer cells in vitro. Methods: Estrogen receptor binding affinity was evaluated in vitro using purified ER-LBD and 3H-estradiol. Transactivation activity was determined in HEK-293 cells. TOR, TAM and their N-desmethyl (NDM), 4-OH, and 4-OH-NDM metabolites were evaluated at up to [10 μM] in Ishikawa endometrial and MCF-7 breast cancer cells. Results: TAM and metabolites had similar binding affinity to TOR analogs. The 4-OH and 4-OH-NDM metabolites bound to ER subtypes with 5-50-fold greater affinity and were 50-100 more efficient at ER transactivation than TOR and TAM. Endoxifen (a CYP2D6 product) exhibited the greatest potency of all compounds tested. Estrogen induced Ishikawa cell growth with maximal effects observed at 0.1 nM. TOR and TAM prevented estrogen-mediated agonist effects with similar activity. TOR and TAM had similar antiestrogen activity against Ishikawa cell growth, however, 4-OH metabolites of TAM and TOR are up to 377-fold more potent than their parent drugs. These data are consistent with in vivo studies in intact rats demonstrating TOR and TAM block estrogenic effects on the uterus. TOR and TAM and metabolites were also antagonists in MCF-7 cells, with the order of antiestrogenic potency being 4-OH and 4-OH-NDM metabolites > TOR/TAM and NDM metabolites. Conclusions: Our results provide mechanistic insight into the potential contributions of the 4-OH and 4-OH-NDM metabolites to TOR and TAM activity and perhaps an explanation for the importance of CYP2D6 metabolism to TAM but not TOR clinical outcomes.

Novel strategy to restore the activity of taxanes: A multicenter phase II study of combination therapy with taxanes and toremifene at 120 mg for advanced/recurrent breast cancer—Kinki Multidisciplinary Breast Oncology Group (KMBOG0612).

e13611 Background: A preclinical study reported that toremifene (TOR) increased the level of an anticancer agent such as doxorubicin and paclitaxel in the breast cancer cell which has acquired resistance to them by the expression of P-glycoprotein. Methods: The subjects were postmenopausal females with advanced/recurrent breast cancer who showed PD after PR or SD to mono therapy with taxanes (paclitaxel or docetaxel) was confirmed. In a phase II prospective study, 120 mg of TOR was administered orally every day after the treatment failure of taxane mono therapy, while continuing the respective taxane regimen. As a primary end point, we assessed the response rate (RR) including clinical benefits. Secondary end points were the time to progression (TTP) and adverse reactions. Results: Twenty-seven patients were registered between November 1, 2006 and October 31, 2010, and 25 patients were analyzed at this time. The median age was 57 years (range: 36-79) and the median ECOG PS was zero (range: 0-1). The RR, clinical benefit (CB) rate, and median TTP were 8.7%, 21.7%, and 12.1 weeks (range: 3.1-69.6), respectively. Moreover, with adding TOR to taxanes, treatment duration could be extended more than 100% in 5 patients (i.e., paclitaxel+TOR 323 days / paclitaxel 288 days = 112%). Adverse reactions to this combination regimen were observed in 8 patients. In 2 cases, grade 2 numbness was noted. However, the other patients showed grade 1 adverse reactions; this therapy was tolerable. Conclusions: Combination therapy with taxanes and TOR at 120 mg was tolerable and useful. The results suggest that TOR may restore the activity of taxanes in taxane refractory advanced/recurrent breast cancer patients.

Relationship of CYP2D6 status and toremifene metabolism.

e13068 Background: Tamoxifen (TAM) is widely used as endocrine therapy for estrogen receptor (ER) positive breast cancer despite a growing controversy regarding its effectiveness in patients with compromised cytochrome P450 2D6 (CYP2D6) activity. Since it is metabolized to active metabolites via CYP2D6, legitimate concerns have been raised over concurrent use of TAM and drugs that are potent inhibitors of CYP2D6 (e.g., SSRI antidepressants). Toremifene (TOR) is a structural analog of TAM that is approved for the treatment of metastatic breast cancer in postmenopausal women with ER positive or unknown tumors. TOR is primarily metabolized by CYP3A4, while CYP2D6 is thought to play a minor role in its metabolism. We examined the role of individual CYP isoforms in the metabolism of TOR and TAM to N-desmethyl (NDM), 4-hydroxy (4-OH) and 4-OH-N-desmethyl (4-OH-NDM) metabolites. Methods: The in vitro metabolism of TOR and TAM was investigated using human liver microsomes and recombinant CYP proteins. Samples were analyzed by LC-MS/MS. Results: N-demethylation is the primary metabolic pathway for both TOR and TAM. Similar levels of N-desmethyl metabolites were formed for both compounds. N-demethylation occurred by CYP2D6, 3A4 and 1A1 but not appreciably by the other six CYP isoforms tested. Specific inhibitors of CYP2D6 and 3A4 activity blocked N-demethylation. The 4‐OH and 4-OH-NDM metabolites of TAM, but not TOR, were observed when the parent drugs were incubated with the CYP2D6 isoenzyme. The formation of both 4-OH-tamoxifen and 4-OH-NDM-tamoxifen (endoxifen) were significantly inhibited by 2D6 inhibitors, including the SSRI antidepressant paroxetine. These results were corroborated by studies using human liver microsomes from poor, intermediate and extensive CYP2D6 metabolizers. Conclusions: Tamoxifen, but not toremifene, is a substrate for 4-hydroxylation by CYP2D6 in human liver microsomes. The metabolism of toremifene is not markedly affected by potent CYP2D6 inhibitors in human liver microsomes. These results suggest that clinical benefit in subjects taking toremifene to treat metastatic breast cancer would not be subject to allelic variation in CYP2D6 status or affected by co-administration of CYP2D6-inhibiting medications.

Inhibition of FOSL1 overexpression in antiestrogen-resistant MCF-7 cells decreases cell growth and increases vacuolization and cell death

Elevated activator protein-1 (AP-1) activity in breast cancer cells has been linked to Tamoxifen (TAM) resistance. Fos-like antigen-1 (FOSL1) is a member of the AP-1 transcription factor and is overexpressed in a variety of human cancers including breast tumors. We have previously established an estrogen-independent and antiestrogen Toremifene (TOR)-resistant subline of MCF-7 breast cancer cells. In these cells, the expression of FOSL1 is upregulated when compared to the parental cells. In the present study, partial inhibition of FOSL1 expression in these cells by small interfering RNA resulted in a marked decrease of cell growth. The inhibition of cell growth paralleled with changes in cell morphology such as increased formation of vacuoles followed by an increase in the number of dead cells. The inhibition of FOSL1 expression in these cells also restored sensitivity to TOR. Our results suggest that chemotherapy targeting overexpression of FOSL1 could be a potent strategy for treating endocrine resistant breast cancers.

P287 The effects of toremifene (TOR) and letrozole (LET) on serum lipids and bone metabolism in postmenopausal patients with estrogen receptor (ER) positive breast cancer – MULTI03 study interim report

P287 The effects of toremifene (TOR) and letrozole (LET) on serum lipids and bone metabolism in postmenopausal patients with estrogen receptor (ER) positive breast cancer – MULTI03 study interim report

PROSPECTIVE STUDY ON THE EFFECT OF TOREMIFENE IN PATIENTS WITH ADJUVANT ANASTROZOLE FAILURE IN POSTMENOPAUSAL BREAST CANCER

Purpose; An endocrine treatment using aromatase inhibitor (AI) is a standard strategy for postmenopausal patients with hormone receptor positive breast cancer. Postmenopausal patients who relapsed during adjuvant AI treatment or progression within a year after completion of AI treatment were prospectively treated with 40 mg/day of toremifene (TOR) in order to examine the effect of the selective estrogen receptor modulator (SERM) in AI-refractory patients. Patients and Methods; Twenty-three postmenopausal breast cancer patients who relapsed during adjuvant AI treatment or relapsed within a year after completion of AI were enrolled in the prospective trial from January 2007 to February 2010. Out of the patients, 14 cases had measurable or evaluable lesions, and the other had only bone metastasis. All patients were treated with toremifene of 40 mg/day. The primary efficacy end point for the trial was clinical response and secondary end point was progression free survival (PFS).Result; The objective response rate in the patients with measurable lesions was 7.1% (1/14) and clinical benefit rate was 28.6%. In the patients with evaluable bone disease, clinical benefit was 44.4%. No serious adverse event was observed except a patient with grade 3 non-hematological toxicity (AST and ALT).Conclusion; The SERM, toremifene (40 mg/day), demonstrated a safe profile and a favorable effect on disease control after adjuvant AI failure.

The Effect of Toremifene on Lipid Metabolism Compared with That of Tamoxifen in vitro

Background: Tamoxifen (TAM) and toremifene (TOR) prevent estrogen from stimulating breast cancer growth and also have agonistic effects in a number of physiological systems. TAM is known to increase intracellular triglyceride, but the action of TOR on lipid metabolism in vitro has not yet been determined. Aim: To compare the effect of TOR on lipid metabolism with that of TAM in vitro, using HepG2 cell lines. Methods: Intracellular concentrations of total cholesterol and triglyceride in HepG2 cells were measured by an enzymatic method after TAM or TOR treatment. Results: Intracellular concentrations of total cholesterol were decreased by both TAM and TOR, but not significantly different from the control level. TOR produced no changes in the intracellular concentrations of triglyceride, whereas TAM increased the intracellular concentrations of triglyceride at concentrations ranging from 10^–7 to 10^–5 mol/l of TAM (p < 0.05). Moreover, significant differences were noted between the two treated groups at concentrations ranging from 10^–9 to 10^–5 mol/l (p < 0.05). Conclusion: TOR treatment did not increase intracellular concentrations of triglyceride, although TAM treatment did so.

655 Preliminary report of efficacy of abiraterone acetate in patients with estrogen (ER) or androgen receptor (AR) positive, advanced breast carcinoma resistant to standard endocrine therapies

Toremifene (TOR) and Tamoxifen (TAM) are widely used as endocrine therapy for estrogen receptor positive breast cancer. Poor metabolizers of TAM are likely to have worse clinical outcomes than patients who exhibit normal TAM metabolism due to lower plasma level of its active metabolite, 4-hydroxy-N-desmethyl (4OH-NDM) tamoxifen (endoxifen). In this study, we examined the role of individual cytochrome P450 (CYP) isoforms in the metabolism of TOR to N-desmethyl (NDM), 4-hydroxy (4OH) and 4OH-NDM metabolites in comparison with TAM using human liver microsomes (HLMs) with selective chemical inhibitors for each CYP isoform and recombinant CYP proteins. Similar levels of NDM metabolites were formed for both TOR and TAM, and N-demethylation of both compounds was primarily carried out by CYP3A4. We found that the formation of 4OH-NDM-TOR was catalyzed both by CYP2C9 and CYP2D6, whereas the formation of 4OH-TAM and endoxifen was specifically catalyzed by CYP2D6 in HLMs. Our results suggest that the potential contribution of CYP2D6 in the bioactivation pathway of TOR may be lower compared to TAM, and may have a different impact on clinical outcome than CYP2D6 polymorphisms.

Transcriptional modulation of BCRP gene to reverse multidrug resistance by toremifene in breast adenocarcinoma cells

Breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette half transporter, confers multidrug resistance (MDR) to a series of antitumor agents such as mitoxantrone, daunorubicin, SN-38, and topotecan, and often limits the efficacy of chemotherapy. Recent studies have indicated that a putative estrogen response element (ERE) is located in the promoter region of the BCRP gene. However, whether and how BCRP is regulated transcriptionally by toremifene (TOR) remains unknown. In the present study, two plasmid vectors have been designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a constitutive cytomegalovirus (CMV) promoter as control, respectively, which were transfected into estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 human breast adenocarcinoma cell lines. We showed that toremifene alone significantly downregulated BCRP mRNA and protein levels in estrogen receptor α (ERα)-positive MCF-7 cells in a dose-dependent manner, and the inhibitory effect was partially reversed by estrone (E(1)). Furthermore, gel shift assays demonstrated that specific binding of ERα to the ERE in the BCRP promoter is essential for transcriptional inhibition of BCRP by toremifene. Interestingly, toremifene alone increased the cellular accumulation of mitoxantrone in BCRP-transfected cells, suggesting that TOR indeed inhibits BCRP-mediated drug efflux and overcome drug resistance. To the best of our knowledge, this is the first report describing a direct effect of toremifene on BCRP. Our results thus indicate that toremifene by itself downregulates BCRP expression to reverse BCRP-mediated atypical multidrug resistance via a novel transcriptionally mechanism, which might be involved in TOR-ER complexes binding to the ERE of BCRP promoter to repress transcription of BCRP gene.

Effect of toremifene and ospemifene, compared to acolbifene, on estrogen-sensitive parameters in rat and human uterine tissues.

Although the first generation selective estrogen receptor modulator (SERM) tamoxifen (TAM) is well known for its uterotrophic activity, this study compares the stimulatory effect of the TAM derivatives toremifene (TORE) and ospemifene (OSPE) on estrogen-sensitive parameters in rat and human uterine tissues. Ovariectomized female rats were treated daily orally for 10days with 0.75mg/rat of TORE, OSPE or acolbifene (ACOL, a pure estrogen antagonist in the uterus and mammary gland), which was used for comparison. Human endometrial carcinoma Ishikawa cells were incubated for 5days with increasing doses of compounds, in the absence or presence of 1nM estradiol (E2). TORE and OSPE revealed 52% and 56% increases, respectively, in uterine weight, whereas ACOL had no effect. Similar effects were observed on vaginal weight. Endometrial epithelial height increased from 15.82±0.20 to 48.94± 2.12 and 42.14±1.95μm with TORE and OSPE, respectively, whereas ACOL had no effect. Alkaline phosphatase activity, an estrogen-sensitive parameter in Ishikawa cells, was increased by 144% and 135% with OH-TORE and OH-OSPE, respectively. Owing to their intrinsic estrogenic activity, at maximal concentrations, OH-TORE and OH-OSPE blocked the stimulatory effect of E2 by only 89% compared to 100% with ACOL. The present in vitro and in vivo data show similar stimulatory effects of 4-hydroxytoremifene (OH-TORE) and OH-OSPE on estrogen-sensitive parameters. ACOL, a third generation SERM, has no effect on any of these parameters. Such data add to the potential uterine safety limitations of triphenylethylene-derived SERMs for long-term use in humans.

Comparing the effects of atamestane, toremifene and tamoxifen alone and in combination, on bone, serum lipids and uterus in ovariectomized rats

Complete estrogen blockade remains under investigation as a means to optimize anti-estrogen therapy in breast cancer thus both the efficacy and end-organ toxicities are of interest with combinations. We hypothesized that a steroidal aromatase inhibitor (AI) atamestane (ATA) alone, and in combination with the anti-estrogens tamoxifen (TAM) or toremifene (TOR) would have beneficial effects in ovariectomized (OVX) rats on key end-organ functions including bone and lipid metabolism and on the endometrium. Significant positive effects on bone were noted with ATA, TOR, TAM, ATA + TOR, or ATA + TAM. TOR, TAM, ATA + TOR, or ATA + TAM caused significant decreases in serum cholesterol and low-density lipoprotein cholesterol whereas ATA had no effect. Uterine weight and epithelium lining height were not increased by ATA but were by TOR and TAM. No significant differences were found in the key parameters outlined above between OVX rats given TOR and ATA + TOR, or TAM and ATA + TAM. Our data show that ATA in combination with TOR or TAM is equivalent to TOR or TAM alone in terms of end-organ effects within a range of clinically relevant doses. Further studies of combinations of AIs with anti-estrogens on end-organ function are merited.

Pharmacokinetic analysis of a combined chemoendocrine treatment with paclitaxel and toremifene for metastatic breast cancer

Multidrug resistance protein could be a target for improving the efficacy of paclitaxel (PXL). Toremifene (TOR) may moderate P-gp-related drug resistance in vitro. Some P-gp moderators may change the pharmacokinetic parameters of PXL in vivo. A pharmacokinetic (PK) study in metastatic breast cancer patients (MBC) was conducted to determine the safety and efficacy of PXL and TOR. Fifteen patients received 80 mg/m(2) PXL (i.v.) weekly and 120 mg/body TOR (p.o.) daily. For the pharmacokinetic study, PXL was administered on days 1, 8, 15, 32, and 39; TOR was given from day 18 to the end of study. On days 1, 8, 15, 18, 32, and 39, blood samples were collected from the patients who received either PXL alone or PXL + TOR, and these were analyzed by high-performance liquid chromatography. Among the 15 patients enrolled in the study, one showed a partial response, and eight had a stable disease. TOR caused no specific adverse events that were greater than grade 3, and its toxicity profile in combination with PXL was similar to that of PXL monotherapy. The PK profile of PXL was similar with or without TOR. The PK parameters of PXL indicated no inter- or intra-patient variability in previously treated patients with MBC. No increased PXL toxicity was observed. The PK profile of combined PXL and TOR was similar to that of PXL monotherapy. The addition of TOR to PXL in previously treated patients with MBC appears safe.

Effect of combined therapy with the antiestrogen agent toremifene and local hyperthermia on breast cancer cells implanted in nude mice

We evaluated the effects of combined treatment with the antiestrogen agent toremifene (TOR) and local hyperthermia (LHT) on the MCF-7 breast cancer cell line. BALB/c mice implanted with MCF-7 cells were divided into six treatment groups: a control group, a TOR30 group (given 30 mg/kg/day), a TOR120 group (given 120 mg/kg/day), an LHT group (43.5 degrees C), a TOR30 + LHT group, and a TOR120 + LHT group. The effects of the treatments on tumor cells, estrogen receptor (ER) expression, and cell cycle kinetics were measured after 21 days. We calculated the apoptotic index and vascular density inside the tumors and evaluated the efficacy of the transmigration of TOR into the tumors. The antitumor effects were significantly greater in both combined therapy groups than in any of the single therapy groups. Estrogen receptor expression was weaker in the combined therapy groups than in the single therapy groups, and there were more G0/G1-phase cells and fewer S-phase cells in both combined therapy groups than in the single therapy groups. The apoptotic index was increased and the tumor vascular density was decreased in the combined therapy groups. We attributed the effects of this combined therapy to the induction of apoptosis, the decrease in vascular density, and the increase and decrease in G0/G1-phase and S-phase cells, respectively, in the tumors.

Rationale for salvage therapy with high-dose selective estrogen receptor modulator after treatment failure of aromatase inhibitors in breast cancer

1129 Background: Aromatase inhibitors (AIs) are currently widely used for hormone responsive breast cancer patients as adjuvant therapy and first line treatment for metastatic breast cancer (MBC). Salvage therapy has become important to the patients failed these primary AIs. We explored the possible application of high-dose selective estrogen modulators (SERMs) in vitro and in vivo. Methods: As a model for treatment failure of AI, MCF-7 cells were cultured in estradiol (E2)-deficient medium for over 6 months. Survived cells were established as MCF-7 long term E2 deprived (LED) clone. Cell counting kit-8 was used for proliferation assay, and transcription activity of Estrogen Receptor (ER) on estrogen-responsive element (ERE) was measured with luciferase assay. HI-FAIR study (n=63) was conducted as a retrospective chart review from nine Japanese institutes for demonstrating the possible efficacy of high-dose toremifene (120mg/day p.o.) after AI treatment failure in MBC patients. Results: In western blotting, MCF-7 LED clone showed increased expression of ER and Her2, and reduction of Progesterone Receptor (PR) expression as compared to parent MCF-7 cell. Although addition of various concentration of E2 did not enhance the proliferation of LED cell, ERE-dependent transcription was still activated by E2 in same degree as parent MCF-7. Of note was 30-fold increase of basal transcriptional activity in LED cell due to phosphorylation of ER. Fulvestrant (FUL), 4-OH Tamoxifen (TAM) and Toremifene (TOR) all inhibited growth of LED cell. IC50 of FUL for parent MCF-7 and LED cell are identical, and maximum inhibition ratio was about 65%. Whereas, TAM and TOR showed more favorable response to LED cell than to parent MCF-7 cell. Growth capability of LED cell was reduced by about 90% by both TAM and TOR in dose dependent manner. In HI-FAIR study high-dose TOR showed 14.3% response rate, 34.9% clinical benefit and 7.4 months median TTF as the first (n=4), second to third (n=40) and fourth (n=19) lines MBC therapy after AI treatment. Conclusions: Although the efficacy of FUL in the patients previously treated with Ais has been already reported, this study demonstrated the possible contribution of high-dose SERM for these patients. No significant financial relationships to disclose.

Pharmacokinetic study of a combination chemo-endocrine treatment of paclitaxel and toremifene

13017 Background: The mechanism of paclitaxel (PXL) resistance has been investigated. P-glycoprotein (P-gp) on cell membrane could be an important target for improving efficacy of PXL. Toremifene (TOR) may moderate P-gp-related drug resistance in vitro. To determine safety and efficacy of PXL and TOR, we conducted a pharmacokinetic study for a combination chemo-endocrine treatment of PXL plus TOR for metastatic breast cancer. Methods: Patients with metastatic breast cancer, who were previously treated with a standard chemotherapy without PXL, were eligible. Patients received PXL 80 mg/m2(i.v.) weekly on days 1, 8, and 15 in a cycle. Concurrently, toremifene 120mg/body (p.o.) was given dairy from day 1–28 in a cycle. For pharmacokinetic study, single agent of PXL was initially administrated on day 1, 8, and 15. From day 18, TOR 120mg/body was given dairy. On day 22, PXL administration in second cycle was skipped. On day 32, blood samples were collected from the patients received PXL 80 mg/m2 + TOR 120 mg/day/body. Samples were analyzed by HPLC (Unisil Q 5CN). Response was evaluated by WHO criteria and adverse events were evaluated by NCI•CTC Ver.2. Results: Nineteen patients were enrolled. Out of 19, 15 patients had measurable disease, and 1 partial response, 8 stable disease, and 6 progressive disease were observed. The addition of TOR caused in no specific adverse events more than grade 3 and toxic profile in combination was similar that in PXL monotherapy. Pharmacokinetic profile of PXL was similar with/without TOR. In addition, Cmax of TOR was 1.85±0.71 mg/mL. Tmax was 2.9±1.8 hr were observed. AUC0–8 of TOR was not affected in the presence of PXL. AUC0- 8 of TOR-1, metabolite of TOR, also demonstrated the similar pharmacokinetic profile. Pharmacokinetic parameters of PXL did not reveal intra-patient variability in previously treated metastatic breast cancer patients. Conclusions: Pharmacokinetic profile in a combination of PXL and TOR was similar that in PXL monotherapy. The addition of TOR to PXL for previously treated metastatic breast cancer patients might be safety. No significant financial relationships to disclose.

Randomized trial of toremifene to reduce high grade prostatic intraepithelial neoplasia (HGPIN) prior to radical prostatectomy (RP)

5150 Background HGPIN may be a precursor lesion for prostate cancer (PCA). The selective estrogen-receptor modulator toremifene (TOR) has shown chemopreventive activity in preclinical models of PCA. The effect of TOR on HGPIN was studied in men with PCA prior to RP. Methods Men with biopsy-proven PCA, scheduled for RP were randomized (2:1) either to TOR 40 mg po qd for 3 to 6 weeks prior to RP or to no therapy. The primary endpoint was the proportion of HGPIN in the RP specimen. Planned sample sizes were 35 and 17 per arm respectively, providing 93% power if treatment increased the proportion of patients with no RP HGPIN from 15% to 35% and decreased the mean log nonzero HGPIN values from 1 to ¼ standard deviation. Expression of bcl2, ki67, and CD31 were determined on biopsy (BX), and RP tissues, separately for normal adjacent to tumor (NAT), HGPIN, and PCA tissues. Serum hormone levels and toxicity were also assessed. Results The treatment arm accrued 31 patients, the control arm 13 patients. RP HGPIN ranged from 0.5% to 12%. There was no difference in HGPIN means between treatment (3.8%) and control (3.3%) (P=0.77 one-sided Wilcoxon) HGPIN at baseline predicted HGPIN at RP (Spearman correlation=0.64, P=0.0005) but 66% of patients had no detected HGPIN at baseline. Bcl2, ki67, and CD31 were mutually positively correlated in all 3 tissues, in both BX and RP specimens. In both BX and RP specimens, bcl2 was highest by far in HGPIN, and lowest in PCA. Ki67 was lowest in NAT, elevated in HGPIN and highest in PCA. CD31 showed no differences. In treated patients but not in controls, dihydrotestosterone, testosterone, androstenedion, and estradiol increased significantly between baseline, day 14 and final. TOR was well tolerated. Conclusions These data do not support an effect of TOR on HGPIN over 3 to 6 weeks at 40 mg po qd. No significant financial relationships to disclose.