Review of the pharmacological properties of toremifene

New compounds were synthesized with the aim to develop new anti-estrogenic antitumor drugs. The biological properties of the molecules were screened by (1) estrogen receptor (ER) binding, (2) effect on MCF-7 cells, (3) uterotrophic effect and inhibition of estradiol induced uterotropic effect and (4) antitumor effect in DMBA induced rat mammary cancer. One of the molecules, Fc-1157a = toremifene, exhibited the following characteristics: competitive inhibition of [3H]estradiol binding to ER (IC50 = 0.3 mumol/l), inhibition of MCF-7 cell growth in a concentration-dependent manner and cell-killing effect at higher than 3 mumol/l concentrations. Minimal estrogenic dose of toremifene on rat uterus weight was about 40 times higher than that of tamoxifen. Toremifene had statistically significant effect against DMBA-induced rat mammary cancer. Further screening consisted of antitumor, pharmacokinetic and safety studies. Toremifene inhibited the growth of ER-negative, glucocorticoid sensitive, mouse uterine sarcoma in a dose-dependent manner. Pharmacokinetics and metabolism of toremifene resembled closely those of tamoxifen, but since the chlorine atom of the toremifene molecule was not metabolically cleaved tamoxifen and toremifene did not have chemically similar metabolites. Toremifene was well tolerated in animal toxicity studies. No hyperplastic or neoplastic nodules, which were seen in almost all high-dose (48 mg/kg for 24 weeks) tamoxifen-treated rats, were found in toremifene-treated rats (dose 48 mg/kg). In clinical phase I studies in healthy voluntary postmenopausal women, no side effects were reported, at doses less than or equal to 460 mg, neither after a single dose nor after five daily doses. At the dose of 680 mg two out of five persons experienced vertigo and headache. Toremifene, at the dose of 68 mg daily, had antiestrogenic effect on estradiol-induced human vaginal epithelial cells. Clinical phase II studies have confirmed that toremifene has a promising antitumor effect.     

Differential binding of antiestrogens by rat uterine and chick oviduct cytosol

Analysis of the interactions of two synthetic estrogen antagonists, tamoxifen and CI 628, with rat uterine and chick oviduct cytosol revealed significant differences in the antiestrogen binding properties of these tissues. In the rat uterus CI 628, tamoxifen and estradiol were bound to a similar number of saturable binding sites and estradiol could completely inhibit the binding of tritiated antiestrogens to these sites. In contrast, high affinity, saturable antiestrogen binding sites in chick oviduct were present at three times the concentration of estradiol binding sites and estradiol could only partially inhibit the binding of tritiated antiestrogens to these sites. It is concluded that antiestrogens bind to the estrogen receptor in both tissues and that chick oviduct has an additional saturable antiestrogen binding site distinct from the classical estrogen receptor site.     

Comparative effects of raloxifene, tamoxifen and estradiol on human osteoblasts in vitro: Estrogen receptor dependent or independent pathways of raloxifene

SERMs bind to both estrogen receptor (ER)α and β, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.     

Direct and reversible inhibition of estradiol-stimulated prolactin synthesis by antiestrogens in vitro

The antiestrogens tamoxifen and monohydroxytamoxifen inhibited the estradiol-stimulated increase in prolactin synthesis by dispersed cells in culture derived from immature rat pituitary glands. Monohydroxytamoxifen had a relative binding affinity for the estrogen receptor similar to that of estradiol, whereas tamoxifen's relative binding affinity was approximately 3%. This was consistent with the observation that monohydroxytamoxifen was 30 times more potent than tamoxifen as an antiestrogen in vitro. To avoid the possibility that tamoxifen was fractionally metabolized to monohydroxytamoxifen by the pituitary cells, the p-methyl, p-chloro, and p-fluoro derivatives of tamoxifen that are unlikely to be converted to monohydroxytamoxifen were tested for activity. The substitution did not have a detrimental effect on their ability to inhibit the binding of [3H]estradiol to either rat uterine or pituitary gland estrogen receptors. Similarly, the derivatives of tamoxifen inhibited estradiol-stimulated prolactin synthesis at concentrations that were consistent with their relative binding affinities. Although it is clearly an advantage for tamoxifen to be metabolized to the more potent antiestrogen monohydroxytamoxifen, we have shown that this is not a prerequisite for the antiestrogenic actions of tamoxifen. With the direct actions of antiestrogens established, the pituitary cell system was validated for further structure-activity relationship studies. Overall, the inhibition of estradiol-stimulated prolactin synthesis by antiestrogens is competitive and reversible with estradiol, an effect that can be explained by interactions with the estrogen receptor system.     

三苯氧胺和雌二醇对大鼠子宫雌激素受体的竞争性结合作用

利用改进的葡聚糖活性炭饱和分析法（ＤＣＣ法）,对卵巢切除大鼠注射雌二醇（Ｅ组）或同时注射雌二醇和三苯氧胺（Ｅ＋Ｔ组）后６ｈ至６４ｄ,进行子宫ＥＲ含量测定,发现,Ｅ＋Ｔ组比Ｅ组的ＥＲ值小并且增加缓慢;Ｅ组的ＥＲ值在注射后３０ｄ达到最大值而且数值超过对照组（Ｃ组）。计算机曲线拟合Ｅ组或Ｅ＋Ｔ组与Ｃ组的ＥＲ比值求得：Ｅ组开始注射雌二醇时ＥＲ水平为Ｃ组的２．０３３倍,即＝２．０３３,恢复过程的时间常数τ＇为５１．５５ｄ,３￣Ｈ－雌二醇与ＥＲ结合过程的时间常数τ为１３．０ｄ;Ｅ＋Ｔ组的＝１．３１５,τ＇＝３８．７６ｄ,τ＝２１．５５ｄ,说明同时给予三苯氧胺和雌二醇情况下,三苯氧胺有抑制雌二醇诱导ＥＲ水平升高的作用,并减缓３￣Ｈ－雌二醇与ＥＲ的结合过程。另外,还求得Ｅ＋Ｔ组中三苯氧胺和ＥＲ和结合率约占４３．７％,表明三苯氧胺与雌二醇对ＥＲ有竞争性结合作用。     

Estrogen- and antiestrogen-induced ornithine decarboxylase activity and uterine growth in the rat

The estrogen antagonists tamoxifen and monohydroxytamoxifen are also classified as partial estrogen agonists. In infantile rats, estradiol induced a single peak of uterine ODC activity at 6h following injection regardless of the extent of induction by various estradiol doses. By contrast, the timing of the ODC activity peak induced by tamoxifen and monohydroxytamoxifen was highly dependent upon the dosing conditions and was delayed to 18 h at lower tamoxifen doses. In immature rats, tamoxifen and monohydroxytamoxifen induced two peaks of uterine ODC activity resembling those induced by estradiol. Both ODC activity peaks were delayed by 9 h, without decreases in peak heights, by a 50-fold tamoxifen dose reduction. In all experiments the initial appearance of antiestrogen- and estradiol-induced ODC activity corresponded to initial uterine wet weight gain regardless of dosing condition. Thus, when dose-related temporal shifts are taken into account, tamoxifen and monohydroxytamoxifen are complete agonists with respect to induction of uterine weight gain and ODC activity.     

Interaction of DP-TAT-59, an active metabolite of new triphenylethylene-derivative (TAT-59), with estrogen receptors.

DP-TAT-59, (Z)-2-(4-(1-(4-hydroxyphenyl)-2-(4-isopropylphenyl)-1-butenyl) phenoxy)-N, N-dimethylethylamine, has been reported to inhibit estrogen-stimulated growth of MCF-7 cells as well as rat uterus at lower concentrations than the hydroxymetabolite of tamoxifen (4-OH-TAM). In the present study, the growth of mouse Leydig cell tumor, B-1F cells were also more effectively inhibited by DP-TAT-59 than 4-OH-TAM. Additionally, the expression of estrogen responsive element ligated CAT gene transfected into B-1F cells was also suppressed by DP-TAT-59. Thus, the interaction of DP-TAT-59 with estrogen receptor (ER) was characterized and compared with that of 4-OH-TAM using immature rat and bovine uteri. The dissociation constant of DP-TAT-59 to ER of immature rat uterus was 0.24 nM and was similar to that of 4-OH-TAM (Kd = 0.20 nM) and estradiol (Kd = 0.29 nM). Using sucrose density gradients, the sedimentation constant of DP-TAT-59 with bovine uterus was 4.9S, which was similar to that of estradiol (5.1S) and 4-OH-TAM (5.3S). However, the elution profile of the DP-TAT-59-ER complex from a DEAE-Sephadex column was different for both estradiol-and 4-OH-TAM-ER complexes. These results suggest that ER forms different complexes with DP-TAT-59 than estradiol or 4-OH-TAM, while the ER binding affinity of these compounds are similar to each other.     

Characterization of estrogen receptors in the hamster brain

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.     

Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Elution of ER was obtained by chelating agents or by imidazole, thus indicating that histidine residues on the ER molecule are involved in the interaction with the metal. Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentrations. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. Furthermore, if added to pretransformed ER-hormone complex, they strongly inhibited the binding of the complex to isolated nuclei. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is therefore speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity

Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer.     

Effect of ribonuclease on the physico-chemical properties of estrogen receptor

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3-4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25 degrees C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.     

Estrogen Receptor Characterization Following Selective Sedimentation Separation of Estrogen-Binding Components In Immature Rat Uterine Cytosol

Abstract

Characterization of a specific estrogen receptor (ER) in fetal and early postnatal rat uterine cytosol is complicated by the presence of other high-affinity estrogen-binding components, such as alpha-fetoprotein (AFP). In an attempt to circumvent their influence, we have employed the selective sedimentation of unlabeled cytosol through sucrose gradients, followed by the analysis of [³H]estradiol binding to a pool of fractions comprising the ER region, as well as to individual gradient fractions. As the amount of AFP present in 21-day-old rats is sufficiently low to permit ER characterization by conventional methodology, we have validated the selective sedimentation method by comparing its results with those obtained conventionally. Though conventional gradient analysis revealed only one estrogen-binding component, saturation and binding inhibition analyses indicated the presence of multiple components, identified as AFP and the ER. These conclusions were supported by results from labeling individual gradient fractions obtained following selective sedimentation of unlabeled cytosol. Further, when unlabeled 7–9 S gradient fractions were pooled and assayed by saturation and binding inhibition analyses, only one binding component, with ER characteristics, was revealed. These results validate selective sedimentation as an effective method for separating multi-component estrogen-binding systems and suggest its applicability to similar systems.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

Effect of neonatal treatment with mifepristone or tamoxifen on the binding capacity of the thymic glucocorticoid or uterine estrogen receptor of adult rats: data on the mechanism of hormonal imprinting

For studying the mechanism of perinatal hormonal imprinting newborn rats were treated with a single injection of the antihormones, mifepristone (RU486) or tamoxifen (100 microg each). Glucocorticoid receptors of thymi of 6 weeks old male and female, and uterine estrogen receptors of 2 months old female rats were studied for dexamethasone or estradiol binding, respectively. Tamoxifen caused faulty imprinting both in the thymic and uterine receptors, increasing affinity and density of males, and decreasing females' glucocorticoid receptors as well, as decreasing the density of uterine estradiol receptors. Neonatal mifepristone treatment was indifferent to the thymus, and decreasing to density of uterine estrogen receptors. Males' body weight significantly decreased 6 weeks after tamoxifen treatment. The results suggest that imprinting can not be provoked by a molecule (hormone antagonist) which can bind to the receptor without any postreceptorial events (mifepristone/glucocorticoid receptor), in the presence of some postreceptorial effects the reaction takes place, however the strongest reaction can be observed by the hormone analogue (tamoxifen) with postreceptorial (agonist) effect, not considering that the receptor is the direct target of the molecule or a cross-reaction is present.     

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.     

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiol's activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.