Tamoxifen is currently the endocrine therapy of choice for early and advanced breast cancer. Attempts to improve the therapeutic efficacy have included altering the triphenylethylene ring structure of tamoxifen, forming
ABSTRACT: Tamoxifen is currently the endocrine therapyof choice for early and advanced breast cancer. Attempts to improve thetherapeutic efficacy have included altering the triphenylethylene ringstructure of tamoxifen, forming new nonsteroidal ring structures or creatingsteroidal estradiol analogs with greater antiestrogenic activity. Thereare now six nonsteroidal compounds either on the market or in clinicaldevelopment and one steroidal "pure" antiestrogen has enteredclinical trials. A number of these agents show improved estrogen-receptorbinding affinity, antiestrogenic activity, and/or antitumor activity comparedwith tamoxifen. Preclinical and clinical data on these compounds are discussedand compared with tamoxifen when possible. [ONCOLOGY 11(Suppl 1):59-64, 1997]
Triphenylethylene structures similar to tamoxifen (Nolvadex) were firstused in the 1940s, but in comparison to synthetic and natural estrogensused at that time, they were not particularly potent antitumor agents.[1,2] Tamoxifen was introduced into the clinic in 1969. Because of itsmuch improved toxicity profile over the estrogens and androgens in useat that time, it rapidly gained acceptance as the endocrine treatment ofchoice for advanced breast cancer and subsequently as adjuvant therapy.[3-5]
Three different strategies have been used to develop new antiestrogens:(1) chemical alteration of the triphenylethylene ring of tamoxifen, (2)production of new nonsteroidal ring structures (eg, the benzothiophenes),and (3) production of steroidal estradiol analogs with pure antiestrogenicactivity (Figure 1).
All of the nonsteroidal antiestrogens discussed in this article arepartial estrogen agonists to differing degrees. In contrast, the steroidalpure antiestrogens lack intrinsic estrogenic activity and appear to havea somewhat different mechanism of action. Although both types of compoundsbind to the estrogen receptor (ER), the tamoxifen analogs allow partialactivation of estrogen receptors, permitting transcription of some estrogen-regulatedgenes, whereas the steroidal antiestrogens appear to completely inhibittranscriptional activity via the estrogen receptor.
The six nonsteroidal antiestrogens shown in Figure1 are either marketed or in clinical trials. All but one, raloxifene,are based on the triphenylethylene molecule. In addition, pure steroidalantiestrogens (ICI 164,384 and ICI 182,780) have been developed in orderto produce a state of complete estrogen deprivation. The overall goal forthe development of new antiestrogens is to increase their efficacy andsafety (Table 1).
This last characteristic is important because many women taking theseagents are treated for long periods with adjuvant therapy. Since most breastcancer patients are unlikely to receive hormone replacement therapy dueto fear of disease recurrence, new endocrine breast cancer agents withan improved side-effect profile and favorable effects on bone mineral densityand the cardiovascular system would be highly valuable.
The purpose of this review is: (1) to describe the current status andultimate potential of the nonsteroidal antiestrogen agents and (2) to introducea new class of pure steroidal antiestrogen agents.
Tamoxifen has gained wide clinical acceptance, first for metastaticbreast cancer and then for adjuvant therapy. Currently, tamoxifen is beingstudied for the chemoprevention of breast cancer in healthy women at highrisk for the disease. The use of tamoxifen has expanded over the yearsbecause of its efficacy in prolonging overall and disease-free survivalas well as reducing the incidence of contralateral breast cancer. Becausetamoxifen is a mixed estrogen agonist and antagonist, it has been shownto have positive, estrogenic effects on bone and the cardiovascular systemwhile maintaining antitumor activity via its antiestrogenic effects. Theestrogenic activity of tamoxifen has been associated with a proliferativeeffect on the endometrium and a two- to threefold increase in the riskof endometrial carcinoma. This association has stimulated interest in thediscovery of new antiestrogens with no uterotropic activity. The methodsfor evaluating these compounds are shown in Table2.
Toremifene (Fareston) differs from tamoxifen by only a single chlorineatom (see Figure 1). Although it producesdifferent metabolites due to the stability of the chlorine atom, toremifenehas a pharmacologic profile similar to tamoxifen.
Toremifene displaced [3H]estradiol binding to estrogen receptorsby 50% at a concentration of 0.5 mmol/L. In the ER-positive human breastcarcinoma cell line MCF-7, toremifene showed a pattern of activity similarto tamoxifen: growth inhibition at low concentrations and oncolytic activityat high concentrations. A concentration of 5 × 10-6M toremifene killed all MCF-7 cells within two days. The activity oftoremifene appears to be estrogen-dependent; growth inhibition of MCF-7cells at concentrations less than 10-6 M can be reversed withestradiol.
Against dimethylbenzanthracene (DMBA)-induced rat mammary cancer, toremifeneand tamoxifen showed similar antitumor activity; the one difference wasthat 45 mg/kg tamoxifen was toxic to the rat, whereas 45 mg/kg toremifeneshowed an antitumor effect.
In the rat uterus, the estrogenic effects of toremifene appear to belower than tamoxifen at low and moderate concentrations; however, the maximumestrogenic response was similar. Toremifene has also demonstrated estrogeniceffects on human endometrial tissue in postmenopausal breast cancer patients,including increasing endometrial thickening and proliferation.
Unlike tamoxifen, toremifene did not produce liver tumors in rats atdoses up to 48 mg/kg for one year.
Toremifene has been studied in a comparative trial with tamoxifen in648 perimenopausal or postmenopausal women with metastatic breast cancer(hormone receptor positive or unknown receptor status). The three treatmentarms consisted of tamoxifen (20 mg/d), toremifene (60 mg/d), or toremifene(200 mg/d). In the intent-to-treat analysis, the frequency of objectiveresponse (complete or partial) was 19% for tamoxifen, 21% for 60-mg toremifene,and 23% for 200-mg toremifene (P not significant between treatments). Themedian response durations were 19.1 months, 16.9 months, and 18.4 months,respectively (P not significant between treatments). Likewise, there wereno significant differences among the three treatments in the median timeto progression or median overall survival.
Overall, the type and frequency of adverse effects were similar amongthe three groups, including the incidence of tumor flare and thromboembolicand cardiac events; however, the 200-mg toremifene dose was associatedwith a greater frequency of elevated aspartate aminotransferase (AST) levelsand nausea.
At this time, there does not appear to be any benefit of toremifeneover tamoxifen. The clinical experience with toremifene will need to beexpanded substantially to determine if there is any benefit of the drugwith respect to rare adverse events such as endometrial cancer.
Droloxifene (3-hydroxytamoxifen) is currently undergoing clinical trialsin advanced breast cancer. Unlike tamoxifen, droloxifene is itself theactive moiety and thus does not require metabolism for activation.Droloxifene shows a higher binding affinity for ER compared with tamoxifen.The IC50 for the displacement of 17-beta-estradiol from ER is1 × 10-8 M. In rats, droloxifene exhibits higher antiestrogenicactivity and lower estrogenic activity than tamoxifen.
Droloxifene is more effective than tamoxifen in inhibiting the growthof ER-positive breast cancer cells, even at therapeutic concentrations(0.1 to 0.4 mM), and its activity is related to ER content. Interestingly,short-term (1-hour) exposure to droloxifene in vitro produced maximum growthinhibition, leading investigators to conclude that the agent may be suitablefor intermittent therapy. Moreover, droloxifene produced a greater antitumoreffect than tamoxifen against DMBA-induced breast tumors in rats. Droloxifene(at doses up to 200 mg/kg/d for 6 months or doses up to 90 mg/kg/d for24 months) is not hepatocarcinogenic in the rat.
A large phase II trial of droloxifene was conducted in centers locatedin Europe, Canada, and Brazil. The purpose of the study was to comparethree daily doses (20 mg, 40 mg, and 100 mg) in postmenopausal women withadvanced breast cancer. Eligible patients had no prior exposure to systemichormonal therapy and had positive or unknown hormone-receptor status; of369 randomized patients, 268 were evaluable for response.
Complete or partial responses were seen in 30% of the 20-mg group, 47%of the 40-mg group, and 44% of the 100-mg group. The differences were significantbetween the 20-mg group and the 40-mg group (P = .02) and between the 20-mggroup and the 100-mg group (P = .04). Half of the responses were seen withinthe first two months of starting treatment.
The median duration of response was 12 months for 20 mg, 15 months for40 mg, and 18 months for 100 mg. Again, the 40-mg (P = .02) and the 100-mg(P = .01) groups had significantly better results than the 20-mg group.
Side effects reported by more than 20% of patients included hot flashes,lassitude, and nausea; the frequency of these effects did not appear tobe dose related. Thromboembolic events did not occur more frequently thanwith other antiestrogens.
Issues that remain to be resolved for droloxifene include its effectson the uterus, bone, and the cardiovascular system as well as its potentialfor cross-resistance with tamoxifen. Droloxifene has been shown to haveestrogenic effects on bone in the rat, and clinically, has produceddecreases in plasma cholesterol without affecting cholesterol synthesis.Although these results look promising, as with toremifene, droloxifene'sultimate clinical profile will only be determined in trials comparing itwith tamoxifen.
Raloxifene is a benzothiophene derivative with a high binding affinityfor ER, reportedly 2.9-fold greater than that of estradiol. In therat, raloxifene has exhibited antiestrogenic activity in the breast anduterus and estrogen agonist activity on bone and lipids.
In contrast to tamoxifen, raloxifene showed a lack of uterotropic effectafter four days of treatment in ovariectomized rats. Near completeantagonism of estrogen-induced uterotropic activity was observed at 1 mg/d.Moreover, three days of raloxifene was able to block the uterotropic actionof estradiol for 10 subsequent days. Raloxifene was unable to reverse theuterotropic response to tamoxifen. Raloxifene dose-dependently inhibitedestrogen-stimulated proliferation of MCF-7 cells.
Sato et al compared the effects of raloxifene and tamoxifen on bone,cholesterol, and the uterus in six-month-old, ovariectomized rats. Theeffect of raloxifene on bone mineral density was dose-dependent, with anED50 of 0.3 mg/kg/d (35 days of treatment), while the ED50 for tamoxifenwas 0.1 mg/kg/d. At doses of 0.1-10 mg/kg, raloxifene dose-dependentlyreduced cholesterol levels to 51% to 62% of ovariectomized controls. Theresults were similar for tamoxifen. In contrast, uterine epithelial thicknessincreased by 250% with tamoxifen therapy, compared with only 60% seen withraloxifene.
Raloxifene (100 mg orally, two times a day) was studied in 14 patientswith disseminated breast cancer refractory to tamoxifen. There wasone minor response in a patient with soft-tissue disease and in five patientswith stable disease. Side effects included hot flashes (n = 4), fatigue(n = 3), leg cramps (n = 1), and mild nausea (n = 3).
Although the antitumor activity of raloxifene needs further evaluation,the drug may have a role in the prevention of osteoporosis in postmenopausalwomen. In this population, raloxifene doses 50 or more mg/d have significantlyreduced total serum and low-density lipoprotein (LDL) cholesterol as wellas serum markers of bone turnover (eg, osteocalcin, alkaline phosphatase).
TAT-59 is a triphenylethylene derivative in early development in Japan.Like tamoxifen, TAT-59 is converted to an active metabolite, 4-OH-TAT-59.[21,22]TAT-59 shows a binding affinity to ER similar to tamoxifen. The IC50for the inhibition of 5 × 10-9 M estradiol binding torat uterine ER was 5.37 × 10-9 M for 4-OH-TAT-59 and 3.63× 10-9 M for 4-OH-tamoxifen.
Tominaga et al compared the activity of 10-6 M concentrationsof TAT-59, droloxifene, toremifene, and tamoxifen against MCF-7 cells incubatedwith 10-8 M estradiol in vitro. The following order of potency(from greatest to least) was reported after both 48 and 120 hours of incubation:TAT-59, droloxifene, tamoxifen, toremifene.
Toko et al reported that TAT-59 was 2.9- to 5.5-fold more potentthan tamoxifen in inhibiting the uterotropic effect of ovariectomy in immaturerats. Against MCF-7 cells transplanted into nude mice, TAT-59, unlike tamoxifen,was able to suppress tumor growth at a dose of 0.1 mg/kg/d (P < .01). However, the effects of both agents were similar at 0.3 mg/kg/d.Compared with tamoxifen, TAT-59 was 10-fold more active against DMBA-inducedmammary tumors in rats and demonstrated more activity against tumors withlow ER content. Preliminary clinical data indicate that TAT-59 hasactivity similar to tamoxifen.
Idoxifene is a triphenylethylene derivative designed to have a greateraffinity for ER and to be more resistant to metabolism than tamoxifen.In vitro, idoxifene shows 2.5-fold greater binding affinity for ER thantamoxifen.
After three days of incubation, 10-6 M idoxifene inhibitedgrowth of MCF-7 cells in vitro by 58.7% compared with 38.7% for the sameconcentration of tamoxifen. Moreover, idoxifene was 1.5-fold more potentthan tamoxifen in inhibiting estradiol-induced proliferation of MCF-7 cells (P = .006). The inhibitory effect of idoxifene was reversible by estradiol,and the drug was not active against an ER-negative cell line. Idoxifene'suterotropic activity is reported to be 1.4-fold lower than that of tamoxifen.
Idoxifene showed similar reduction in tumor volume as with tamoxifenin the N-nitrosomethylurea-induced rat mammary cancer model, but was activein a greater percentage of rats (92% vs 75%).
A phase I study of idoxifene was conducted in 20 postmenopausal womenwith advanced breast cancer. Groups of five patients each receivedeither 10, 20, 40, or 60 mg idoxifene daily for two weeks. However, somepatients continued to receive the drug at a dosage of 20 mg/d until diseaseprogression occurred.
Idoxifene was associated with significant reductions in luteinizinghormone (LH) and follicle-stimulating hormone (FSH) levels with no differenceobserved in estradiol and sex hormone binding globulin (SHBG) levels. Fourteenpatients experienced side effects that were not dose related. These effectsincluded mild nausea, anorexia, vomiting, and fatigue. In 14 patients whoremained on the drug for up to 384 days, there were two partial responsesand four patients with stable disease lasting 1.5 to 14 months.
As idoxifene is in the early stages of clinical development, it willbe interesting to see how its antitumor efficacy compares with that oftamoxifen in more advanced clinical trials.
Pure steroidal antiestrogens have been developed by Alan Wakeling andJean Bowler at Zeneca Pharmaceuticals that lack the intrinsic estrogenicactivity of the tamoxifen analogs (see Figure1). Instead of altering the triphenylethylene ring, these researchersdecided to work with the estradiol molecule itself to try to design a potent,steroidal antiestrogen. The Zeneca investigators added a long alkyl sidechain at the 7-alpha position of the B ring of the steroid.
The first candidate, ICI 164,384, was shown to be a potent antiestrogen.However, it was hydrophobic and thus showed reduced bioavailability. ICI164,384 was modified by attaching fluorine atoms to the alkyl side chain.The new compound, ICI 182,780, has affinity for the ER equivalent to thatof estradiol and has now entered clinical development.
In vitro, ICI 182,780 displaced 17-beta-[3H]estradiol bindingto the ER with an IC50 of 0.94 × 10-8 M.
ICI 182,780 dose-dependently blocked the uterotropic effect of estradiolin immature female rats; complete estrogen antagonism was observed at adose of 0.5 mg/kg/d subcutaneously. Peroral administration of ICI 182,780reduced its efficacy by one order of magnitude.
In vitro, ICI 182,780 inhibited the growth of MCF-7 cells with an IC50of 0.29 nM. When the activity of ICI 182,780 was compared with tamoxifenagainst this cell line after three to five days of treatment, only 7% ofcells exposed to 10 nM ICI 182,780 were capable of cell division, comparedwith 37% of those exposed to 4 mM tamoxifen. The concentrationstested were considered optimally antiestrogenic but not cytotoxic.
Against MCF-7 cells xenografted into nude mice, a single 5-mg subcutaneousinjection of ICI 182,780 was equivalent to the antitumor efficacy of fourweeks of daily tamoxifen (10 mg/kg/d orally).
ICI 182,780 has been effective against tamoxifen-resistant human breastcancer cell lines,[28,29] indicating a lack of cross-resistance with tamoxifenand suggesting the possibility for its use as second-line therapy aftertamoxifen failure.
The effects of ICI 182,780 on the human endometrium were studied in30 premenopausal women requiring hysterectomy for benign conditions.ICI 182,780 (12 mg/d intramuscularly) or no treatment was given for sevendays prior to surgery. Treated patients had significantly lower ER levelsin myometrial cells and reduced Ki67 (proliferation-associated nuclearantigen) in the endometria. The treatment had no effect on progesteronereceptor (PR) levels.
In another study using the same dosage and duration, endometrialproliferation was measured using ultrasound during the follicular phaseof the menstrual cycle prior to hysterectomy. In contrast to the controlgroup, treated patients showed no increase in endometrial thickness throughoutthe study, indicating a potent antiestrogenic effect of ICI 182,780 onthe endometrium.
A clinical trial of ICI 182,780 was conducted in 56 postmenopausal womenwith primary breast cancer. Patients were given either no treatment(n = 19) or ICI 182,780 6 mg (n = 21) or 18 mg (n = 16) for seven daysprior to primary breast surgery. Treatment had no significant effect onserum levels of LH, FSH, or SHBG. Compared with pretreatment levels, ICI182,780 produced significant reductions in the expression of ER (P < .01), PR(P < .05), and Ki67 (P < .05) in ER-positivebreast tumors. When the data on Ki67 were separated by dose, the reductionwas only significant for the 18-mg group. ICI 182,780 also reduced expressionof an estrogen-regulated protein (p52) (P < .05). Adverse effects,primarily headaches, were reported by five patients receiving 6 mg andthree patients receiving 18 mg ICI 182,780, and most effects were consideredunrelated to treatment. The injections were well tolerated, with only onelocal reaction.
A phase I study of ICI 182,780 was conducted in 19 patients with advancedbreast cancer resistant to tamoxifen. The first four patients received100 mg as a monthly intramuscular. injection, increased to 250 mg fromthe second month onward. The remaining 15 patients received 250 mg duringthe entire study. Patients were treated until progression occurred; themedian duration was more than 18 months. Thirteen patients responded totreatment (seven partial responses and six with stable disease). Therewere no serious drug-related adverse events and no reports of new hot flashes,vaginal dryness, or altered libido during the study.
In summary, ICI 182,780 is a potent, pure antiestrogen lacking the partialestrogen agonist activity of the nonsteroidal antiestrogens like tamoxifen.ICI 182,780 lacks proliferative activity on the endometrium, suggestingan advantage over tamoxifen with respect to the development of endometrialcancer. The lack of cross-resistance with tamoxifen seen so far indicatesthat tamoxifen-resistant tumors would be likely to respond to second-linetherapy with pure antiestrogens.
There are currently a variety of antiestrogens in development, includingtriphenylethylene derivatives, a benzothiophene derivative, and pure steroidalantiestrogens. New antiestrogens will have to compete with the long-establishedtrack record of tamoxifen, including its reduced recurrence of breast cancerand mortality rates as well as its favorable side-effect profile.
The activity of toremifene in vitro and in vivo appears to be very similarto that of tamoxifen, and no apparent benefit to the new agent is seenat this time. A number of candidates, including droloxifene, TAT-59, idoxifene,and ICI 182,780, have demonstrated good preclinical and clinical activityand will need to be tested in phase III trials against tamoxifen. Althoughraloxifene exhibits favorable ER-binding affinity and estrogen antagonistactivity, published data on its antitumor activity are lacking. Raloxifenemay prove to be a successful therapy for the prevention of osteoporosis.
All of the investigational drugs discussed in this article require furtherevaluation. However, it should be noted that even after nearly 20 yearsof clinical use, the full clinical potential of tamoxifen may not yet betotally realized. Trials of intermittent and alternating tamoxifen therapyneed to be conducted to determine whether tamoxifen resistance can be avoided.
For the new compounds, an evaluation of their potential for cross-resistancewith tamoxifen as well as their effects on the endometrium, bone mineraldensity, and lipid profiles is essential. Only after these data becomeavailable will the ultimate potential of these agents be evident.
1. Haddow A, Watkinson JM, Patterson E: Influence of synthetic oestrogensupon advanced malignant disease. Br Med J 2:393-398, 1944.
2. Walpole AL , Patterson E: Synthetic oestrogens in mammary cancer.Lancet 2:783-786, 1949.
3. Cole MP, Jones CTA, Todd IDH: A new anti-oestrogenic agent in latebreast cancer: and early clinical appraisal of ICI 146474. Br J Cancer25:270-275, 1971.
4. Ward HWC: Anti-oestrogen therapy for breast cancer: a trial of tamoxifenat two dose levels. Br Med J 1:13-14, 1973.
5. Early Breast Cancer Trialists' Collaborative Group: Systemic treatmentof early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomisedtrials involving 31,000 recurrences and 24,000 deaths among 75,000 women.Lancet 339:1-15; 71-85, 1992.
6. Howell A, Downey S, Anderson E: New endocrine therapies for breastcancer. Eur J Cancer 32A:576-588, 1996.
7. Kangas L: Review of the pharmacological properties of toremifene.J Steroid Biochem 36:191-195, 1990.
8. Kangas L, Nieminen AL, Blanco G, et al: A new triphenylethylene compound,Fc-1157a. II. Antitumor effects. Cancer Chemother Pharmacol 17:109-113,1986.
9. Tomas E, Kauppila A, Blanco G, et al: Comparison between the effectsof tamoxifen and toremifene on the uterus in postmenopausal breast cancerpatients. Gynecol Oncol 59:261-266, 1995.
10. Hayes DF, Van Zyl JA, Hacking A, et al: Randomized comparison oftamoxifen and two separate doses of toremifene in postmenopausal patientswith metastatic breast cancer. J Clin Oncol 13:2256-2566, 1995.
11. Pritchard KI: Summary. Am J Clin Oncol 14(suppl 2):S62-63, 1991.
12. Hasmann M, Rattel B, Loser R: Preclinical data for droloxifene.Cancer Lett 84:101-116, 1994.
13. Eppenberger U, Wosikowski K, Kung W: Pharmacologic and biologicproperties of droloxifene, a new antiestrogen. Am J Clin Oncol 14 (suppl2):S5-S14, 1991.
14. Rauschning W and Pritchard KI: Droloxifene, a new antiestrogen:Its role in metastatic breast cancer. Breast Cancer Res Treat 31:83-94,1994.
15. Zhu Ke H, Simmons HA, Pirie CM, et al: Droloxifene, a new estrogenantagonist/agonist, prevents bone loss in ovariectomized rats. Endocrinology136:2435-2441, 1995.
16. Lindenthal B, Mezger J, von Bergmann K: Tamoxifen and droloxifenelower plasma cholesterol concentrations in postmenopausal women with breastcancer by different mechanisms (abstract). Naunyn-Schmiedeberg's ArchivPharmacol 353(suppl):R163, 1996.
17. Black LJ, Jones CD, Falcone JF: Antagonism of estrogen action witha new benzothiophene derived antiestrogen. Life Sci 32:1031-1036, 1983.
18. Fuchs-Young R, Glasebrook AL, Short LL, et al: Raloxifene is a tissue-selectiveagonist/antagonist that functions through the estrogen receptor. Ann NYAcad Sci 761:355-360, 1995.
19. Sato M, Rippy MK, Bryant HU: Raloxifene, tamoxifen, nafoxidine,or estrogen effects on reproductive and nonreproductive tissues in ovariectomizedrats. FASEB J 10:905-912, 1996.
20. Buzdar AU, Marcus C, Holmes F, et al: Phase II evaluation of Ly156758in metastatic breast cancer. Oncology 45:344-345, 1988.
21. Toko T, Matsuo KI, Shibata J, et al: Interaction of DP-TAT-59, anactive metabolite of new triphenylethylene-derivative (TAT-59), with estrogenreceptors. J Steroid Biochem Mol Biol 43:507-514, 1992.
22. Toko T, Sugimoto Y, Matsuo KI, et al: TAT-59, a new triphenylethylenederivative with antitumor activity against hormone-dependent tumors. EurJ Cancer 26:397-404, 1990.
23. Tominaga T, Yoshida Y, Matsumoto A, et al: Effects of tamoxifenand the derivative (TAT) on cell cycle of MCF-7 in vitro. Anticancer Res13:661-666, 1993.
24. Chander SK, McCague R, Luqmani Y, et al: Pyrrolidino-4-iodotamoxifenand 4-iodotamoxifen, new analogues of the antiestrogen tamoxifen for thetreatment of breast cancer. Cancer Res 51:5851-5858, 1991.
25. Coombes RC, Haynes BP, Dowsett M, et al: Idoxifene: Report of aphase I study in patients with metastatic breast cancer. Cancer Res 55:1070-1074,1995.
26. Parker MG, Arbuckle N, Dauvois S, et al: Structure and functionof the estrogen receptor. Ann NY Acad Sci 684:119-126, 1993.
27. Wakeling AE, Dukes M, Bowler J: A potent specific pure antiestrogenwith clinical potential. Cancer Res 51:3867-3873, 1991.
28. Coopman P, Garcia M, Brunner N, et al: Anti-proliferative and anti-estrogeniceffects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifen-resistant humanbreast cancer cells. Int J Cancer 56:295-300, 1994.
29. Lykkesfeldt AE, Madsen MW, Briand P: Altered expression of estrogen-regulatedgenes in a tamoxifen-resistant and ICI 164,384 and ICI 182,780 sensitivehuman breast cancer cell line, MCF-7/TAMR-1. Cancer Res 54:1587-1595, 1994.
30. Dowsett M, Howell R, Salter J, et al: Effects of the pure anti-oestrogenICI 182780 on oestrogen receptors, progesterone receptors and Ki67 antigenin human endometrium in vivo. Human Reprod 10:262-267, 1995.
31. Thomas EJ, Walton PL, Thomas NM, et al: The effects of ICI 182,780,a pure anti-oestrogen, on the hypothalamic-pituitary-gonadal axis and onendometrial proliferation in pre-menopausal women. Human Reprod 9:1991-1996,1994.
32. DeFriend DJ, Howell A, Nicholson RI, et al: Investigation of a newpure antiestrogen (ICI 182780) in women with primary breast cancer. CancerRes 54:408-414, 1994.
33. Howell A, DeFriend D, Robertson J, et al: Response to a specificantioestrogen (ICI 182780) in tamoxifen-resistant breast cancer. Lancet345:29-30, 1995.