Initiating activity of the anti-estrogen tamoxifen, but not toremifene in rat liver

A striking difference between two structurally related anti-estrogen medicines is that tamoxifen is strongly hepatocarcinogenic in the rat, whereas toremifene lacks such activity. To study the basis for this difference, the initiating potential of tamoxifen and toremifene were studied by measurement of rapid induction of hepatocellular altered foci (HAF) that express placental-type glutathione S-transferase in the livers of female Sprague-Dawley (S-D) rats and female Fischer 344 (F344) rats. Both agents were administered by gavage at equimolar doses up to a dose that produced marked weight gain suppression. In rats given the high dose of 40 mg/kg per day tamoxifen continuously for 36 weeks, 75% of S-D rats developed liver neoplasms, in contrast to only 10% of F344 rats. In the S-D strain, tamoxifen produced a tendency to increased HAF at 2 weeks at the dose of 40 mg/kg per day and by 12 weeks, a dose-related increase was evident. In contrast, toremifene induced no HAF even at the equimolar high dose of 42.4 mg/kg per day for 12 weeks. The induction of HAF by tamoxifen was less in the F344 rats. Neither agent elicited increases in hepatocellular proliferation in S-D or F344 rats. When phenobarbital was administered for 24 weeks as a promoting agent after the anti-estrogens, S-D rats given tamoxifen at 20 mg/kg per day for 12 weeks, developed liver neoplasms, but not F344 rats or rats of either strain given even a higher dose (42.4 mg/kg) of toremifene. Thus, tamoxifen has initiating activity in these rat strains whereas toremifene does not.      

DNA adducts caused by tamoxifen and toremifene in human microsomal system and lymphocytes in vitro

DNA-binding of tamoxifen and toremifene was studied in rat invivo, in human and rat microsomes in vitro, and in culturedprimary human lymphocytes by ³²P-postlabelling. Only tamoxifencaused DNA adducts in rat liver. Both compounds induced adductsin both rat and human micro-somal systems and in cultured lymphocytes.The levels of adducts in microsomes and lymphocytes were low,ca. 1 adduct/10⁹ nucleotides. Toremifene showed lower bindingin each system, and in lymhocytes adducts were only detectedat a cytotoxic dose level.     

Clastogenic and aneugenic effects of tamoxifen and some of its analogues in hepatocytes from dosed rats and in human lymphoblastoid cells transfected with human P450 cDNAs (MCL-5 cells)

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4- hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4- hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4- hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.      

Peroxidase activation of tamoxifen and toremifene resulting in DNA damage and covalently bound protein adducts

When [¹⁴C]tamoxifen was incubated with horseradish peroxidaseand H₂O2, two major metabolites, separated and identified byHPLC, were N-desmethyltamoxifen and tamoxifen N-oxide. Toremifeneincubated in a similar system yielded N-desmethyltoremifeneand toremifene N-oxide. No 4-hydroxylated metabolites were detectedwith either drug. When calf thymus DNA was included in peroxidaseincubation mixtures, DNA damage, as assessed by ³²P-postlabelling,could also be detected. The extent of damage caused by tamoxifenand toremifene was similar. The major adducts formed followingincubation of DNA with tamoxifen had similar R_f values to twoof the ³²P-postlabelled adducts seen following dosing of ratswith tamoxifen. Peroxidase was able to activate both drugs toderivatives which covalently bound to bovine serum albumin.The pH optimum for covalent binding and N-demethylation wasnear to pH 6.0. Results from liquid chromatography-electrospraysecondary ion mass spectrometry suggest that tamoxifen and toremifeneare metabolized by peroxidase to putative reactive epoxide intermediatesresponsible for the genotoxic effects. It is proposed that peroxidaseoxidizes tamoxifen to a carbon-centred free radical which reactswith oxygen to form peroxy radicals capable of inserting anoxygen atom into tamoxifen. Lactoperoxidase and prostaglandinsynthase are also able to catalyse tamoxifen N-demethylationand binding to protein. These data show that peroxidase canactivate both tamoxifen and toremifene to an intermediate(s)that can damage DNA and covalently react with protein. Sinceit is known that women treated with tamoxifen can develop endometrialtumours, it may be relevant to determine whether activationof tamoxifen by peroxidases may contribute to its carcinogenicaction at extrahepatic sites.     

Delayed effects of tamoxifen in hepatocarcinogenesis-resistant Fischer 344 rats as compared with susceptible strains

The anti-oestrogenic drug tamoxifen has been under investigation as a breast cancer chemopreventive agent for at least a decade. However, its use for this purpose is still debatable since it is able to induce liver tumours in rats via a mechanism involving metabolic activation to a DNA adduct-forming electrophilic intermediate. The metabolic activation and adduct-forming properties of tamoxifen are now well characterized but less is known about its ability to induce hepatic cell proliferation, which is also essential for the carcinogenic process. The effects of tamoxifen on liver weight and cell proliferation were compared in female Fischer 344 (F344), Wistar and Lewis rats given the drug in the diet for up to 26 weeks. The onset and duration of hepatic cell proliferation varied between the strains of rat. In Wistar and Lewis but not F344 rats there was a marked increase in hepatocellular proliferation during the first 4 weeks of tamoxifen administration. In the Wistar strain this was associated with an increase in DNA adduct levels; no such increase was observed in the F344 strain. The onset of the proliferative response was delayed until the 13 week time point in the F344 strain. By the 13 and 26 week time points, cell proliferation in tamoxifen-treated Wistar and Lewis rat liver had returned to normal, but the amount of apoptotic activity in these livers was elevated. This suggests that excess cells generated during the proliferative phase of tamoxifen treatment were being eliminated by apoptosis. In the F344 strain, however, increased proliferative activity was associated with relatively low apoptotic activity at the 26 week time point, suggesting that the delayed proliferative response had yet to be balanced by apoptotic deletion. This is consistent with the fact that tamoxifen-induced hepatocellular tumours develop very late, towards the end of the lifespan, in this strain. The cell proliferative activity of tamoxifen in the Wistar rat liver was compared with that of a non-mutagenic analogue, toremifene. Tamoxifen induced increased cell cycle activity in the livers of rats following gavage dosing at all sampling times (1-12 weeks), whereas toremifene had no effect on the incidence of cycling in hepatic cells, demonstrating that the hepatic cell proliferation is not a general response to anti-oestrogen treatment. These observations suggest that the rate of promotion of liver tumours by tamoxifen is a function of the rate, time of onset and duration of increased cell replication. The susceptibility of rat strains to the hepatocarcinogenic effects of tamoxifen appears to depend upon the balance between initiation via DNA adduct formation, promotion via increased cell proliferation and cell deletion via apoptosis. Our findings suggest that an early proliferative response to tamoxifen is important in this process.     

Comparison of the effects of tamoxifen and toremifene on liver and kidney tumor promotion in female rats

Female rats were subjected to a 70% partial hepatectomy andadministered either diethylnitrosamine (10 mg/kg) or the solvent,trioctanoin. After a 2 day recovery from the surgery, the ratswere placed on basal diet alone or containing phenobarbital(500 mg/kg diet), mestranol (0.2 mg/kg diet), tamoxifen (250or 500 mg/kg diet) or toremifene (250, 500 or 750 mg/kg diet)for 6 or 18 months prior to killing. The livers and kidneyswere prepared for pathological diagnoses. In addition, sectionsof liver from the 6 month killing were frozen and serially sectioned.The sections were stained for expression of the placental isozymeof glutathione S-transferase (GST), gamma glutamyl transpeptidase(GGT), canalicular ATPase (ATP) and glucose 6-phosphatase (G6P)and scored by quantitative stereology for number and volumefraction of liver occupied by altered hepatic foci (AHF) withalterations in these markers individually and combined (ANY).Each of the agents increased the volume fraction of liver occupiedby AHF when the ANY category was used. Statistical increasesin both the GGT-positive and G6P-deficient AHF populations wereobserved in the spontaneously as well as DEN-initiated groupstreated with tamoxifen or toremifene. After 18 months of administration,the highest concentration of tamoxifen increased the incidenceof malignant hepatic neoplasms in non-DEN-initiated rats. Toremifene,at the highest tested dose, increased the incidence of hepatocellularcarcinomas in the DEN-initiated groups to a level one-thirdthat observed with tamoxifen administration to DEN-initiatedrats. Both tamoxifen and toremifene increased the incidenceof hypernephromas in previously DEN-initiated rats. While bothtamoxifen and toremifene are effective promoting agents forDEN-initiated lesions, tamoxifen is more potent than toremifenein the induction of rat hepatocarcinogenesis.     

Effect of long-term tamoxifen exposure on genotoxic and epigenetic changes in rat liver: implications for tamoxifen-induced hepatocarcinogenesis

Tamoxifen is a non-steroidal anti-estrogen used for the treatment of breast cancer and, more recently, as a chemopreventive agent in healthy women at high risk of developing breast cancer. On the other hand, tamoxifen is a potent hepatocarcinogen in rats, with both tumor-initiating and tumor-promoting properties. There is substantial evidence that hepatic tumors in rats are initiated as a result of formation of tamoxifen-DNA adducts; however, events subsequent to DNA adduct formation are not clear. Recently, it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations. In the current study, we investigated whether or not the mechanism of tamoxifen-induced hepatocarcinogenesis includes both genotoxic and epigenetic components. Female Fisher 344 rats were fed a 420 p.p.m. tamoxifen diet for 6, 12, 18 or 24 weeks. Hepatic tamoxifen-DNA adduct levels, as assessed by high-performance liquid chromatography and electrospray tandem mass spectrometry, were 580 adducts/10(8) nt at 6 weeks, and increased to approximately 1700 adducts/10(8) nt by 18 weeks. Global liver DNA hypomethylation, as determined by an HpaII-based cytosine extension assay, was increased at all time points, with the maximum increase (approximately 200%) occurring at 6 weeks. Protein expressions of maintenance (DNMT1) DNA methyltransferase and de novo DNA methyltransferases DNMT3a and DNMT3b were decreased at all time points. Likewise, trimethylation of histone H4 lysine 20 was significantly decreased at all time points. In contrast, non-target tissues (i.e. mammary gland, pancreas and spleen) did not show any changes in global DNA methylation or DNA methyltransferase activity. These data indicate the importance of genotoxic and epigenetic alterations in the etiology of tamoxifen-induced hepatocarcinogenesis.     

Genotoxicity of tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s

The clastogenicity of tamoxifen and toremifene was tested insix human lymphoblastoid cell lines each expressing increasedmonooxygenase activity associated with a specific transfectedhuman cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 orCYP3A4). The chemicals were also tested in a cell line (MCL-5)expressing elevated native CYP1A1 and containing transfectedCYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, andin a cell line containing only the viral vector (Ho1). Dose-relatedincreases in micronuclel were observed when cells expressing2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. Thepositive responses in the cell lines were in the order MCL-5> 2E1 > 3A4 > 2D6. Toremifene also gave positive resultswith 2E1, 3A4 and MCL-5 cells, although the responses were lessmarked and the positive effects required higher doses than withtamoxifen. A synthesized epoxide of tamoxifen was also testedin these cell lines and produced similar increases in the incidencesof micronucleated cells. The increases in the responses observedwith the epoxide were greater than with tamoxifen or toremifene.The P450 isoenzyme activities in these cells were in a rangesimilar to those of human tumour-derived cell lines. Microsomes(1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells allmetabolized tamoxifen. The major metabolite detected by HPLCwas N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detectedin cells with cytochrome P450 2E1 and 2D6. These results areconsistent with the following conclusions. (1) Tamoxifen requiresmetabolic activation to DNA-reactive species by specific CYPmonooxygenases in order to exert its genotoxic effects. (2)The positive clastogenic effects elicited in lymphoblastoidcells by tamoxifen epoxide suggest that the genotoxic (and possiblythe carcinogenic) effects of tamoxifen may be due to one ormore epoxide metabolites that are generated intracellularly,probably in close proximity to the nudeus. (3) Tamoxifen ismore genotoxic than toremifene.     

三苯氧胺与脂肪性肝病

三苯氧胺用于雌激素受体阳性乳腺癌患者的内分泌治疗效果肯定,但长期应用可诱发脂肪性肝病.其机制包括药物聚集于肝细胞线粒体、脂肪酸氧化异常、雌激素拮抗作用等.对服用三苯氧胺的乳腺癌患者需定期监测肝功能、血脂、超声及CT等.可选择托瑞米芬、芳香化酶抑制剂替代治疗及应用降脂药物等进行防治.     

三苯氧胺与脂肪性肝病

三苯氧胺用于雌激素受体阳性乳腺癌患者的内分泌治疗效果肯定，但长期应用可诱发脂肪性肝病。其机制包括药物聚集于肝细胞线粒体、脂肪酸氧化异常、雌激素拮抗作用等。对服用三苯氧胺的乳腺癌患者需定期监测肝功能、血脂、超声及CT等。可选择托瑞米芬、芳香化酶抑制剂替代治疗及应用降脂药物等进行防治。     

The tamoxifen dilemma.

The anti-oestrogen tamoxifen is widely used for adjuvant therapy in the treatment of women with breast cancer and has a low incidence of serious side-effects. It could also play a role as a breast cancer chemopreventive agent. However, epidemiological studies in both tamoxifen-treated breast cancer patients and in healthy women have shown that treatment results in a small increase in the incidence of endometrial cancers. While the use of tamoxifen in breast cancer patients is clearly justified, the situation for its use as a chemopreventive agent in healthy women is not so clear cut. Reasons for caution come from studies in rats that show that tamoxifen is a genotoxic mutagenic liver carcinogen. Initiation of tumours in the rat is the result of metabolic activation of tamoxifen by CYP enzymes to an electrophile(s) that binds irreversibly to DNA. This is not related to the oestrogen receptor status of the tissue. The extent of DNA damage, detected by 32P-post-labelling or accelerator mass spectrometry, is dependent both on the dose and the length of exposure. Studies have been carried out to see if such binding occurs in the uterine endometrium from tamoxifen-treated women. Results are presently inconclusive, but if such irreversible DNA binding occurs, it is at very low levels. Based on a mechanistic understanding of tamoxifen-induced liver carcinogenesis in the rat, it seems that in humans hepatic DNA damage will be close to the limit of detection by 32P-post-labelling and liver cancer will not be a significant carcinogenic risk. We cannot be certain of the mode of action of tamoxifen that results in the increase in endometrial cancers in treated women but it seems unlikely that this will be associated with a classical genotoxic mechanism.     

Tamoxifen induces endometrial and vaginal cancer in rats in the absence of endometrial hyperplasia

Tamoxifen was administered orally to neonatal rats on days 2-5 after birth and the subsequent effects on the uterus were characterized, morphometrically, over the following 12 months. Tamoxifen inhibited development of the uterus and glands in the endometrium, indicating a classical oestrogen antagonist action. Between 24 and 35 months after tamoxifen treatment there was a significant increase in the incidence (26%) of uterine adenocarcinomas and a 9% incidence of squamous cell carcinomas of the vagina/cervix in the absence of any oestrogen agonist effect in the uterus. This demonstrates that an oestrogen agonist effect is not an absolute requirement for the carcinogenic effect of tamoxifen in the reproductive tract of the rat. The unopposed oestrogen agonist effect of tamoxifen on the endometrium may not be the only factor involved in the development of endometrial cancers. It is possible that tamoxifen causes these tumours via a genotoxic mechanism similar to that seen in rat liver. However, using (32)P-post-labelling we failed to find evidence of tamoxifen-induced DNA adducts in the uterus. Tamoxifen may affect hormonal imprinting of oestrogen receptor responses in stem cells of the uterus, causing reproductive tract cancers to arise at a later time, in the same way as has been proposed for diethylstilbestrol. If these rodent data extrapolate to humans, then women who are taking tamoxifen as a chemopreventative may have an increased risk of vaginal/cervical cancer, as well as endometrial cancer.     

Inhibition by pentachlorophenol of the initiating and promoting activities of 1'-hydroxysafrole for the formation of enzyme-altered foci and tumors in rat liver

The hepatocarcinogen 1'-hydroxysafrole (HOS) exhibited weakinitiating activity and strong promoting activity for the inductionof enzyme-altered foci and tumors in rat liver. Thus, administrationof a single dose of HOS to rats 18 h after a 70% hepatectomy,followed by administration of phenobar-bital (PB) in the dietfor 6 months, induced a low, but statistically significant,number of foci of enzyme-altered cells. This treatment did notresult in gross liver tumors, even when the PB treatment wascontinued for 16 months. Large numbers of enzyme-altered focideveloped when HOS was administered in the diet at levels of0.05–0.25% to rats previously administered a single doseof N,N-diethylnitros-amine (DEN) 24 h after a 70% hepatectomy.Similarly, rats given a single dose of DEN 24 h after a partialhepatectomy and then fed 0.10 or 0.25% of HOS in the diet for10 months developed a high incidence of hepatocellular carcinomas.In the absence of pretreatment with DEN, dietary administrationfor at least 4 months of 0.10 or 0.25% of HOS induced significantnumbers of enzyme-altered foci; these data and liver tumor inductionby continuous feeding of HOS, in the absence of pretreatmentwith DEN, provide additional evidence for an initiating, aswell as a promoting, activity of HOS in rat liver. Concurrentadministration of the hepatic sulfotransferase inhibitor pentachlorophenolwith HOS in each of the above assays almost completely inhibitedthe initiating and promoting activities of HOS for the formationof enzyme-altered foci and tumors; these data strongly suggestthat both the initiating and promoting activities are mediatedby the sulfuric acid ester, 1'-sulfooxysafrole. HOS also exhibitedinitiating activity in adult mouse liver. Thus, dietary administrationof 0.25% of HOS for only 1 month, followed by administrationof the hepatic tumor promoter 1,4-bis[2-(3,5-dichloropyridyloxy)]benzeneresulted in a high incidence and multiplicity of hepatomas by10 months. In the absence of the promoter, administration ofHOS for only 1 month induced no hepatomas; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzenealone induced only a low incidence. In mice not given the promoter,continuous administration of HOS     

Prospective study on gynaecological effects of two antioestrogens tamoxifen and toremifene in postmenopausal women

To assess and compare the gynaecological consequences of the use of 2 antioestrogens we examined 167 postmenopausal breast cancer patients before and during the use of either tamoxifen (20 mg/day, n = 84) or toremifene (40 mg/day, n = 83) as an adjuvant treatment of stage II-III breast cancer. Detailed interview concerning menopausal symptoms, pelvic examination including transvaginal sonography (TVS) and collection of endometrial sample were performed at baseline and at 6, 12, 24 and 36 months of treatment. In a subgroup of 30 women (15 using tamoxifen and 15 toremifene) pulsatility index (PI) in an uterine artery was measured before and at 6 and 12 months of treatment. The mean (+/-SD) follow-up time was 2.3 +/- 0.8 years. 35% of the patients complained of vasomotor symptoms before the start of the trial. This rate increased to 60.0% during the first year of the trial, being similar among patients using tamoxifen (57.1%) and toremifene (62.7%). Vaginal dryness, which was present in 6.0% at baseline, increased during the use of tamoxifen (26.2%) and toremifene (24.1%). Endometrial thickness increased from baseline (3.9 +/- 2.7 mm) to 6.8 +/- 4.2 mm at 6 months (P< 0.001), and no difference emerged between the 2 regimens in this regard. Before the start of the antioestrogen regimen, the endometrium was atrophic in 71 (75.5%) and proliferative in 19 of 94 (20.2%) samples; 4 patients had benign endometrial polyps. During the use of antioestrogen altogether 339 endometrial samples were taken (159 in tamoxifen group, 180 in toremifene group). The endometrium was proliferative more often in the tamoxifen group (47.8%) than in the toremifene group (32.2%) (P< 0.0001). 20 patients had a total of 24 polyps (17 in tamoxifen and 9 in toremifene group, P< 0.05) during the use of antioestrogens. One patient in the toremifene group developed endometrial adenocarcinoma at 12 months, and one patient had breast cancer metastasis on the endometrium. Tamoxifen failed to affect the PI in the uterine artery, but toremifene reduced it by 15.0% (P< 0.05) by 12 months. In conclusion, tamoxifen and toremifene cause similarly vasomotor and vaginal symptoms. Neither regimen led to the development of premalignant endometrial changes. Our data suggest that so close endometrial surveillance as used in our study may not be mandatory during the first 3 years of use of antioestrogen treatment.     

Phase II trials with toremifene in advanced breast cancer: A review

The antitumor activity of the new triphenylethylene drug toremifene has been studied in advanced breast cancer of postmenopausal women as first line treatment at dose levels of 20, 60, and 240 mg, and as second line or later treatment at high dose levels of 200–240 mg. The response rates (complete + partial response) have been 21% with 20 mg (14 patients), 52% with 60 mg (93 patients in three separate trials), and 68% with 240 mg (38 patients) as first line treatment. After failure on previous therapy (hormonal or chemotherapy) the response rates have been about 10% with 200 mg of toremifene (71 patients in two different trials). In patients whose disease had previously responded to tamoxifen with at least stabilization, the response rate with toremifene has been 23%; but among unselected patients, including patients progressing during adjuvant tamoxifen, the response rate (CR + PR) with toremifene in tamoxifen failures has been 3%. If long lasting (more than 5 months) stabilization of the disease is also considered, a further 20% of previously treated patients have benefitted from toremifene. The treatment has been well tolerated at all dose levels. The most reported side effects have been hot flushes (8–19%) and nausea (8%). 0–6% of patients in different trials have interrupted the treatment because of side effects     

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.     

High dose toremifene in advanced breast cancer resistant to or relapsed during tamoxifen treatment

Summary Fifty patients with advanced breast cancer refractory to prior tamoxifen therapy were assigned to investigational treatment with high-dose toremifene administered 120 mg orally twice a day. Treatment was generally well tolerated. The majority (80%) of the patients had no side effects, and among the remaining 10 patients reported side effects were mostly mild and/or transient. Two objective tumor responses were observed: one complete response (CR), duration 6.2 months, and one partial response (PR), duration 8 months. The response rate was thus 4% (95% CI: 0.5 to 14%). In addition 3 patients experienced a mixed response, some metastatic sites responding, while at other sites disease progressed; 22 patients had disease stabilization for > 2 months. A subset analysis disclosed that a small subgroup of patients, including 7 patients in this study, who had achieved CR at some of the sites during preceding tamoxifen therapy, experienced a long progressionfree time during high dose toremifene treatment. The median time to progression in this subgroup of patients was 9.4 months (95% CI: 3.8 to 9.4) as opposed to 2.1 months (95% CI: 2.0 to 2.8) for all the remaining 43 patients, which is a significant decrease in disease progression (p < 0.03). Such results reveal that although this kind of second-line hormonal treatment with high dose toremifene cannot be recommended for all tamoxifen failures, there might be a subset of patients, i.e. those who achieve CR in some lesion during tamoxifen therapy, who benefit from this type of treatment.     

A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes

The metabolisms of tamoxifen in female rat, mouse and humanliver microsomal preparations were compared. Rat, mouse andhuman liver microsomes were incubated with tamoxifen in thepresence of NADPH and MgCl₂ and the metabolites formed wereanalysed by on-line HPLC electrospray ionization MS. The majormetabolites formed by rat liver microsomes were 4-hydroxytamoxifen,4'-hydroxytamoxifen, N-desmethyltamoxifen and tamoxifen N-oxide.In addition, two epoxide metabolites, 3,4-epoxytamoxifen and3',4'-epoxytamoxifen, and their hydrolysed derivatives, 3,4-dihydrodihydroxytamoxifenand 3',4'-dihydrodihydroxytamoxifen, have been identified. Thepattern of the main metabolites obtained with human liver microsomesresembles qualitatively that of rat liver microsomes. The majordifferences between rat and human liver microsomes were thatthe amount of hydroxylated metabolites were much lower in humanand only traces of 3,4-epoxytamoxifen and the correspondingdihydrodihydroxy derivative were detected. No 3',4'-epoxytamoxifendetected in human liver microsomes. The four major metaboliteswere also formed in much larger amounts and with faster ratesof formation by mouse liver microsomes, though tamoxifen N-oxideclearly predominated in this species. Polar metabolites, 3,4-dihydroxytamoxifenand 4-hydroxytamoxifen N-oxide, which were undetectable in ratand human, were formed in significant amounts in mouse microsomes.As in human microsomes, there was only one epoxide metabolite,3,4-epoxytamoxifen, produced by mouse liver microsomes at levelslower than that found in rat. The faster rate of metabolismand the production of polar metabolites may indicate the abilityof mouse to detoxify tamoxifen by rapid elimination comparedwith rat and human. The production of a larger amount of potentiallyreactive epoxide metabolites in rat may be responsible for theliver carcinogenesis in this species.     

Cell Proliferation in Dimethylbenz(A)Anthracene(Dmba)- Induced rat Mammary Carcinoma Treated with Antiestrogen Toremifene

Cell proliferation during antiestrogen toremifene treatment was studied using the DMBA-induced rat mammary carcinoma model. The volume corrected mitotic index (M/V INDEX) and the S-phase fraction (SPF) determined by flow cytometry (FCM) were used as proliferation markers. Two series of rats (A and B) treated with two dose levels of toremifene were used. The two series of tumors appeared to have different growth properties. In series A the tumors were rapidly growing with high proliferation rate. In this series, toremifene (3 mg/kg for 4 weeks) reduced significantly the mean MV/INDEX, but the slight reduction of the mean SPF was not significant. In series B the tumors grew slowly and had low levels of proliferation markers. One-third of the tumors were spontaneously stable in the untreated group. Higher dose of toremifene was used in this series (12 mg/kg for 4 weeks), and the number of regressing or stable tumors was 58% compared with 31% in series A. Taking into consideration the high number of spontaneously stable tumors in series B, it may be concluded that about one-third of the tumors regressed or remained stable due to toremifene treatment in both series. The reduction of the M/V INDEX was significant only when the regressing treated tumors were compared with the growing controls. The reduction of the SPF was not significant. We think that the M/V INDEX is a more appropriate method to measure cell proliferation than is the SPF in this tumor model, where the tumors are heterogenous and, e.g., spontaneous apoptosis is known to be frequent.